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FACULDADE DE BIOCIÊNCIAS
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PPGBCM
AVALIAÇÃO IN VITRO DA CITOTOXICIDADE DO FORMOCRESOL,
DO TRICRESOL FORMALINA E DO FORMALDEÍDO EM TRÊS
DIFERENTES LINHAGENS CELULARES
Melissa Isabel Thomas
ORIENTADORA
Dra. Maria Antonieta Lopes de Souza
CO-ORIENTADORA
Dra. Virgínia Minghelli Schmitt
Porto Alegre
2006
Di
ssertação apresentada ao
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Biologia Celular e Molecular da
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que me guiaram, protegeram, encorajaram e apoiaram.
Com sincera gratidão, alegria e prazer agradeço aos meus
ANJOS visíveis:
• Meus pais, Guido e Teresinha, pelo dom da vida, pela
dedicação ímpar, pelo amor infinito;
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Bruna e Graziele, pelo apoio, pelo estímulo, pela confiança
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A vocês dedico este trabalho, o meu amor e carinho, e agradeço
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PENSAMENTOS
“Meu filho, tudo o que fizeres, faz com doçura, e
mais que a estima dos homens, ganharás o afeto.”
(Eclesiástico, 3, 19)
“...não haveria frutos se as flores não caíssem.”
4
ÍNDICE
LISTA DE ABREVIATURAS.....................................................................................5
RESUMO..................................................................................................................6
APRESENTAÇÃO DO TEMA...................................................................................7
Citotoxicidade dos materiais……………………………….................................7
Testes de Citotoxicidade……………………………………................................7
Ação antimicrobiana do formocresol e do tricresol formalina…………........9
Formocresol……………………………………………...........................................9
Tricresol formalina……………………………………………..............................11
Formaldeído……………………………………………………..............................11
OBJETIVOS...........................................................................................................13
REFERÊNCIAS……………………………………………………………...................14
ARTIGO CIENTÍFICO: revista a ser submetido……………………………………...17
EVALUATION IN VITRO OF FORMALDEHYDE, FORMOCRESOL AND
TRICRESOL FORMALIN CYTOTOXICITY IN THREE DIFFERENT
ESTABLISHED CELL LINES……………………….….…………………………….…18
Abstract…………………………………………………………...............................19
Introduction…………………………….…….……………………………................20
Materials and Methods………………………………………………………….......22
Cell culture.....………………………………………………………………............22
Formaldehyde, formocresol and tricresol formalin in vitro cytotoxicity test:.....22
MTT Assay ....…………………..….………....…………………….……………...23
Statistical analysis……………………………………………………………………24
Results…………………………………………......…………………….…................25
Discussion…………………………………………..………………….…………......26
References………….……………………………………………………………........32
Tables and Figures..……………………………..…….……………………….........35
CONSIDERAÇÕES FINAIS………………………………………………………........42
ANEXOS……………………………………………………………………..……..........44
5
LISTA DE ABREVIATURAS
d - dias
DMEM – Meio essencial mínimo modificado por Dulbecco
DMSO – Dimetil sulfóxido
DNA – Ácido Desoxirribonucléico
DPBS – Solução tampão salina fosfato modificada por Dulbecco
EDTA – Ácido dietilaminotetracético
FA – Formaldeído
FC – Formocresol
h - hora
HeLa – Linhagem Celular Estabelecida (células epiteliais originárias de câncer
cervical humano)
Hep2 – Linhagem Celular Estabelecida (células epiteliais originárias de carcinoma
de laringe humano)
mg - miligrama
mL – mililitro
mM - milimolar
MTT – brometo de 3-(4,5-dimetilazol-2-il)-2,5-difenil tetrazolio
NIH3T3 – Linhagem Celular Estabelecida (Fibroblastos de camundongo)
SFB – Soro fetal bovino
t – tempo
TC – Tricresol formalina
µg- micrograma
µL - microlitro
6
RESUMO
A avaliação da citotoxicidade dos materiais dentais é de grande importância
na Odontologia. O formocresol (FC) e o tricresol formalina (TC) são duas
medicações utilizadas na odontologia para desinfecção do canal radicular, que
contém formaldeído na sua formulação. Estes produtos são considerados bons
antimicrobianos, mas pesquisas a respeito da toxicidade destes medicamentos
ainda são necessárias.
O presente estudo avaliou in vitro a citotoxicidade do formaldeído, do FC e
do TC, utilizando três linhagens celulares estabelecidas, as células HeLa, NIH3T3
e Hep2, cultivadas em condições padrão. As células foram deixadas em contato
com cada produto durante 1, 2, 3, 4 ou 5 minutos, sendo então incubadas por 24
horas, 48 horas ou 7 dias. O teste de citotoxicidade utilizado foi o ensaio MTT.
Os resultados deste estudo mostraram que os produtos testados foram
tóxicos às diferentes linhagens celulares estabelecidas, em todas as condições
testadas. O formocresol foi o produto que apresentou menor citotoxicidade, sendo
este resultado estatisticamente significante quando comparado com o tricresol
formalina e o formaldeído.
7
APRESENTAÇÃO DO TEMA
Citotoxicidade dos materiais
Numerosos e variados agentes químicos são usados como anti-sépticos
tópicos na prática dental. Como eles são administrados diretamente na cavidade
oral, estes agentes deveriam ter baixa toxicidade e alta segurança para a saúde
do paciente [1].
A necessidade da utilização de materiais biocompatíveis na Odontologia
implica que sejam realizados testes de citotoxicidade. A toxicidade de um material
dental pode ser avaliada por testes in vitro, testes in vivo, incluindo
experimentação em animais, e estudos clínicos em humanos. Estudos in vitro são
principalmente realizados para avaliar a citotoxicidade ou a genotoxicidade de um
material dental [2].
A biocompatibilidade pode ser definida como a capacidade de um material
de exercer sua função, na aplicação específica, na presença de uma resposta
apropriada do hospedeiro. Uma reação adversa pode ser devido à toxicidade de
um material dental ou a outros fatores, como a acumulação de bactérias sobre os
materiais, causando inflamações. Portanto, a toxicidade pode ser considerada
como somente uma reação de não compatibilidade de um material dental [2].
Testes de citotoxicidade
As vantagens dos testes de toxicidade in vitro, quando comparado com
experimentos em animais e estudos clínicos em humanos, é o controle das
8
condições experimentais, baixos custos e rápida realização, além de não envolver
questões éticas [2].
Os testes in vitro podem ser realizados utilizando linhagens celulares
permanentes ou culturas primárias (por exemplo, gengiva, mucosa e fibroblastos
de polpa) [3]. Alguns autores afirmam que as culturas primárias refletiriam de
forma mais precisa as situações in vivo, porém apresentam dificuldades no cultivo
[2, 3]. Outros, afirmam que o uso de linhagens celulares estabelecidas oferece
vantagens no cultivo, pois as condições de cultura são definidas, evitando as
variações individuais e a interferência do complexo mecanismo homeostático que
ocorre in vivo [4, 5].
O Comitê Europeu para Padronização (CEN) é o órgão competente que
avalia e recomenda normas para a utilização de materiais biológicos, incluindo os
empregados na odontologia. Este Comitê recomenda o desenvolvimento e uso de
métodos in vitro que possam ser adotados como padrões, minimizando a
necessidade de avaliações in vivo [6].
A viabilidade celular pode ser avaliada através de vários métodos, porém é
aconselhável que a pesquisa utilize um processo que envolva menor tempo e
menor variação na análise das amostras [7].
O ensaio MTT é um teste usado para avaliar a viabilidade celular, de
execução rápida e objetiva, baseado em uma reação colorimétrica. O sal MTT
(brometo de 3-(4,5-dimetilazol-2-il)-2,5-difenil tetrazólio) entra na mitocôndria da
célula viável e é clivado pela enzima succinato desidrogenase, produzindo cristais
formazan, de coloração azul escuro. A quantidade de cristais formada é
9
diretamente proporcional ao número de células viáveis. Assim, quanto mais escura
a coloração ao final da reação, maior é a viabilidade celular. A densidade óptica
resultante do teste MTT é determinada em espectrofotômetro [7].
Ação antimicrobiana do formocresol e do tricresol formalina
A principal proposta do tratamento de canal radicular de dentes com
necrose pulpar é eliminar as bactérias e seus produtos, responsáveis pela
inflamação local, antes de obturar o conduto radicular (Figura 1). Os
microorganismos têm um papel fundamental na etiologia das doenças periapicais
e pulpares [8], e seu controle e eliminação são importantes durante o tratamento
endodôntico [9].
A eliminação dos microorganismos do canal radicular é almejada usando
soluções irrigadoras durante a instrumentação e usando medicações intracanais.
É esperado que estes tratamentos possam atingir as ramificações do canal
radicular e outras áreas inacessíveis ao clínico, auxiliando no tratamento
endodôntico (tratamento de canal) [10].
Formocresol
Nas últimas cinco décadas, a substância mais amplamente usada e aceita
no tratamento de dentes primários, na pulpotomia (tratamento conservador da
polpa radicular dos dentes decíduos), tem sido o formocresol. Estudos têm
demonstrado uma propriedade de fixação tecidual adequada, proporcionando
sucesso radiográfico e clínico [11, 12]. Porém, muito interesse tem surgido sobre o
10
potencial mutagênico e carcinogênico de produtos contendo formaldeído, dos
efeitos tóxicos do formocresol, e da possível difusão desta substância para os
tecidos sistêmicos e circunvizinhos do dente. Em contraste com outros aldeídos, o
formaldeído é uma molécula pequena que penetra através do canal radicular.
Estudos em animais têm confirmado que o formocresol aparece sistemicamente
após seu uso em pulpotomias e que injúria celular pode ocorrer nos tecidos
sistêmicos [13].
O agente ideal da pulpotomia deveria exercer efeito máximo sobre os
microorganismos locais, enquanto sua citotoxicidade deveria ser mínima sobre a
polpa remanescente e os tecidos circunvizinhos. A quantidade de tempo
necessária para a concentração antimicrobiana matar os microorganismos é
também clinicamente relevante. É de pequena importância para o clínico o uso de
concentrações específicas da substância se sua ação não puder ocorrer numa
quantidade de tempo clinicamente razoável (2 – 3 dias). Além disso, é importante
que a substância exiba uma atividade antimicrobiana no local a ser desinfectado
[14].
Atualmente, na rotina endodôntica, o formocresol é usado por 5 minutos na
aplicação clínica. No estudo de Hill, 1991, o formocresol foi usado a 1,5%, e o
tempo máximo necessário para matar todos os microorganismos foi de 2 minutos.
Portanto, uma aplicação de 5 minutos de formocresol foi mais longa que o
necessário para matar os microorganismos em um dente cariado [14].
O formocresol é um agente antibacteriano efetivo quando testado contra
bactérias anaeróbias selecionadas (Peptococcus magnus, Proprionibacterium
acnes, Veillonella parvula, Lactobacillus fermentum, Porphyromonas gingivalis e
11
Fusobacterium nucleatum). Quando comparado com outros medicamentos, o
formocresol produziu significantemente largas zonas de inibição sobre as
bactérias. Pesquisas fortalecem que o formocresol tem demonstrado boas
propriedades antimicrobianas sobre bactérias anaeróbias facultativas [15, 16].
Tricresol formalina
O tricresol formalina é usado como curativo de demora na câmara pulpar de
dentes permanentes com necrose pulpar e preparo químico-mecânico incompleto
do canal. Este medicamento propicia a desinfecção do canal, dando condições
para reparar as lesões periapicais. Ocorre também uma ação bactericida à
distância, pela liberação de vapores do formaldeído [17].
O tricresol formalina é um composto à base de formaldeído e cresol. O
formaldeído é um gás produzido pela incompleta combustão do metanol, é solúvel
em água, apresentando solução aquosa de aproximadamente 38% a 40% de
formaldeído em peso, chamado formalina [18]. O tricresol diminui as propriedades
irritantes do formaldeído. Este material é derivado do “carvão de breu”, sendo um
potente anti-séptico e considerado menos tóxico que o formaldeído [12].
Formaldeído
O formaldeído é um reagente muito reativo, e é conhecido como
potencialmente citotóxico, mutagênico e carcinogênico [19].
Estudo in vitro realizado em células epiteliais da mucosa bucal humana
sugeriu que o formaldeído tem a capacidade de causar numerosos efeitos
citopatológicos [20].
12
A exposição da mucosa oral ao formaldeído pode ocorrer através da
liberação de certos materiais dentais e por inalação pela boca [21]. Uma das
formas de inalação do formaldeído é através de vários materiais odontológicos,
como ionômero de vidro, materiais de base de dentadura acrílica, pastas e
cimentos endodônticos [22].
O risco aparente dos efeitos adversos sobre a saúde quando da exposição
ao formaldeído e pela possibilidade de efeitos potencialmente tóxicos sobre o
tecido oral, através de materiais dentários, tem preocupado o profissional da
odontologia. A citotoxicidade do formaldeído foi investigada no estudo de Nilsson
et al. [20], por meio de cultura de células orais humanas, e o resultado demonstrou
que o formaldeído foi mais tóxico sobre os fibroblastos do que sobre as células
epiteliais [20].
13
OBJETIVOS
Geral
O objetivo deste estudo foi avaliar a citotoxicidade do formocresol, do
tricresol formalina e do formaldeído.
Específicos
• Avaliar a citotoxicidade do formocresol, do tricresol formalina e do formaldeído
em diferentes tempos de exposição (1, 2, 3, 4, e 5 minutos), nas linhagens
celulares:
Hep2 (células epiteliais originárias de carcinoma de laringe humano)
NIH3T3 (fibroblastos de camundongos)
HeLa (células epiteliais originárias de câncer cervical humano)
• Avaliar o efeito citotóxico do formocresol, do tricresol formalina e do
formaldeído após 24 horas, 48 horas e 7 dias de incubação após o tratamento
com os produtos citados, nas linhagens celulares Hep2, NIH3T3, HeLa
14
REFERÊNCIAS
1. GALLOWAY, S. M.; Aardema, M. J.; Ishidate, M. Jr; Ivett, J. L.; Kirkland, D. J.;
Morita, T.; et al. Report from working group on in vitro tests for chromosomal
aberration. Mutat Res, v. 312, p. 241-61, 1994.
2. SCHMALZ, G. Use of cell cultures for toxicity testing of dental materials-
advantages and limitations. J Dent Suppl 2, v. 22, p. S6-S11, 1994.
3. ARENHOLT-BINDSLEV, D.; Bleeg, H. Characterization of two types of human
oral fibroblasts with a potential application to cellular toxicity studies: tooth pulp
fibroblasts and buccal mucosa fibroblasts. Int Endod J, v. 23, p. 84-91, 1990.
4. RIBEIRO, D. A.; Scolastici, C.; Lima, P. L. A.; Marques, M. E. A.; Salvadori, M.
F. Genotoxicity of antimicrobial endodontics compounds by single cell gel (comet)
assay in Chinese hamster ovary (CHO) cells. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod, v. 99, p. 637-40, 2005.
5. EISKJAER, M., Arenhlt-Bindslev D. Cytotoxicity of formaldehyde in cultures of
three different human cell types. J Dent Res, v. 73, p. 952, 1994.
6. SCHMALZ, G.; Browne, R. M. The biological evaluation of medical devices used
in dentistry. Intern Dental J, v. 45, p. 275-278, 1995.
7. MOSMANN, T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunological Methods, v.
65, p. 55-63, 1983.
15
8. KAKEHASHI, S.; Stanley, H. R.; Fitzgerald, R. J. The effects of surgical
exposures of dental pulps in germfree and conventional laboratory rats. Oral Surg
Oral Med Oral Pathol, v. 20, p. 340-9, 1965.
9. VALERA, M. C.; Rego, J. M.; Jorge, A. O. C. Effects of sodium hypochlorite and
five intracanal medications on Cândida albicans in root canals. J Endod, v. 27, p.
401-8, 2001.
10. GOMES, B. P. F. A.; Lilley, J. D.; Drucker, D. B. Variation in the susceptibilities
of components of the endodontic microflora of biomechanical procedures. Int
Endod J, v. 29, p. 235-41, 1996.
11. FUKS, A. B.; Bimstein, E.; Bruchim, A. Radiographic and histologic evaluation
of the effects of two concentration of formocresol on pulpotomized primary and
young permanent teeth in monkeys. Peditr Dent, v. 5, p. 9-13, 1983.
12. ‘S-GRAVENMADE, E. J. Some biochemical consideration of fixation in
endodontics. J Endod, v. 1, p. 233-237, 1975.
13. PASHLEY, E. L.; Myers, D. R.; Pashley, D. H.; Whitford, G. M. Systemic
distribution of
14
C-formaldehyde from formocresol-treated pulpotomy sites. J Dent
Res, v. 5, p. 603-8, 1980.
14. HILL S. D.; Berry, C. W.; Seale, N. S.; Kaga, M. Comparison of antimicrobial
and cytotoxic effects of glutaraldehyde and formocresol. Oral Surg Oral Med Oral
Pathol, v. 71, p. 89-95, 1991.
15. OHARA, P.; Torabinejad, M.; Kettering, J. D. Antibacterial effects of various
endodontic medicaments on selected anaerobic bacteria. J Endod, v. 19, p. 498-
500, 1993.
16
16. VANDER WALL, G. L.; Dowson, J.; Shipman, C. Antibacterial efficacy and
cytotoxicity of three endodontic drugs. Oral Surg, v. 33, p. 230-241, 1972.
17. SOUZA, V.; Holland, R.; Nery, M. J.; Mello, W. Emprego de medicamentos no
interior dos canais radiculares. Ação tópica e à distância de algumas drogas. ARS
Curandi Odontol, p. 4-15, 1978.
18. SIQUEIRA Jr, J. F.; Lopes, H. P. Endodontia: Biologia e Técnica. Rio de
Janeiro: Medsi, p. 397-426, 1999.
19. LEWIS, B. B.; Chestner, S. B. Formaldehyde in dentistry: a review of
mutagênico and carcinogenic potential. JADA, v. 103, p. 429-434, 1981.
20. NILSSON, J. A.; Zheng, X.; Sundqvist, K.; Liu, Y.; Atzori, L.; Elfwing, A.;
Arvidson, K.; Grafström, R. C. Toxicity of formaldehyde to human oral fibroblasts
and epithelial cells: influences of culture conditions and role of thiol status. J Dent
Res, v. 77, p. 1896-1903, 1998.
21. GRAFSTROM, R.C. In vitro studies of aldehyde effects related to human
respiratory carcinogenesis. Mutat Res, v. 238, p. 175 – 84, 1990
22. GEURTSEN, W.; Leyhausen, G. Biological aspects of root canal filling
materials – histocompatibility, cytotoxicity, and mutagenicity. Clinical Oral
Investigations, v. 1, p. 5-11, 1997.
17
ARTIGO CIENTÍFICO
O artigo científico será submetido ao periódico Journal of Endodontics –
QUALIS A – área CB-1 da CAPES.
18
SCIENTIFIC ARTICLE
Evaluation In Vitro of Formaldehyde, Formocresol and Tricresol Formalin
Cytotoxicity in Three Different Established Cell Lines.
Melissa L. Thomas
1
, Virgínia M.Schmitt
1,2
, Tatiana Gonçalves da Silveira
1
, Maria
A. L. de Souza
3
Mail address: Virginia Minghelli Schmitt, PhD
Pontifícia Universidade Católica do Rio Grande do Sul
Faculdade de Farmácia
Avenida Ipiranga, 6681, prédio 12C
CEP 90.619-900, Porto Alegre, RS, Brazil
Phone (+55) (51) 3320-3512; Fax (+55) (51) 3320-3612
e-mail: [email protected]
1
Molecular Biology Laboratory, Instituto de Pesquisas Biomédicas; Pontifícia Universidade Católica
do Rio Grande do Sul (PUCRS), Avenida Ipiranga, 6690, Porto Alegre, RS, Brazil
2
Faculdade de Farmácia, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS),
Avenida Ipiranga, 6681, Pd 12, Porto Alegre, RS, Brazil
3
Micromorphology Laboratory, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS),
Faculdade de Biociências, Avenida Ipiranga, 6681, Pd 12, Bloco C, Porto Alegre, RS, Brazil
19
ABSTRACT
The cytotoxicity evaluation of dental material is of great importance in
dentistry. Formocresol (FC) and tricresol formalin (TC) are two medicines used in
dentistry to disinfect the root canal, both containing formaldehyde (FA) in their
formulation. These products are considered good antimicrobial agents, but
determine the toxicity of these products is still necessary. The present study
evaluates the cytotoxicity in vitro of FA, FC and TC, using three established line
cells, HeLa, NIH3T3 and Hep2, cultivated under standard conditions. Cells were
left in contact with each product during 1, 2, 3, 4 or 5 minutes, being after that
incubated for 24 hours, 48 hours or 7 days. The cytotoxicity test used was the MTT
assay. Our results showed that the test products were toxic to the different cell
lines used, in all assayed conditions. Formocresol was the product which
presented the lowest cytotoxicity, with a statically significant result when compared
with tricresol formalin and formaldehyde.
Key words: cytotoxicity, cell culture, formocresol, formaldehyde, tricresol formalin.
20
INTRODUCTION
A great number of dental materials remain in contact with oral tissues for a
long period of time. The properties and the long exposure of these dental materials
to the oral cavity can be important factors concerning toxicity. For this reason,
biochemical assays to evaluate the cytotoxic effect of these dental materials are
relevant, considering the potential risk for both patient and clinician [1].
The endodontics materials evaluated in this study, formocresol (FC) and
tricresol formalin (TC), have an extent history of use. These materials have a
strong antimicrobial activity on disinfecting the root canal [2, 3]. However, literature
data regarding their cytotoxic effect is still relatively modest when compared to data
on antibacterial effects.
International standards for preclinical in vitro screening of dental materials
allow the use of relevant primary cells; however, the use of established cell lines is
recommended [4]. The use of primary cells does not assure that the observed
results will be reproductive, once primary cells may have specific metabolic
potentials, which does not occur in permanent cell lines [5].
The well established toxic and carcinogenic potentials of formaldehyde have
aroused great interest in dentistry. The knowledge that some dental materials can
liberate formaldehyde created the need of evaluating the toxicity degree of dental
materials [6, 7].
The aim of this study was to evaluate the toxicity of formaldehyde,
formocresol and tricresol formalin in three different established cell lines, exposed
21
to the products in different time intervals, and kept afterwards in different
incubation times. The cytotoxic effect was evaluated by measuring cell viability.
22
MATERIALS AND METHODS
Cell Culture
The established cell lines used were NIH3T3 (mouse fibroblasts), Hep2
(epithelial cells from human larynx carcinoma) and HeLa (human cervical cancer
epithelial cells) (American Type Culture Collection, Rockville, MD).
Cell lines were cultivated in 25cm
2
culture flasks (TPP, Switzerland,
Europe), with Dulbecco Modified Eagle’s Medium (DMEM) (GIBCO, Grand Island,
NY), containing 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY) and
gentamycin (10µg/mL) (GIBCO, Grand Island, NY), and were incubated at 37°C, in
5% CO
2
atmosphere (SANYO MCO-15A, Japan).
Formaldehyde, formocresol and tricresol formalin in vitro cytotoxicity test
Cytotoxicity of formaldehyde (FA) (Nuclear, Diadema, SP, Brazil),
formocresol (FC) (Biodinâmica, Ibiporã, PR, Brazil) and tricresol formalin (TC)
(Diodinâmica, Ibiporã, PR, Brazil) was tested in NIH3T3, Hep2 and HeLa cells
(Table 1). The formaldehyde was diluted in DPBS (Dubecco’s phosphate-buffered
saline solution) (GIBCO, Grand Island, NY) to a concentration of 19%, in order to
be used in the same concentration as it is found in the original formulation of the
test products.
Cells were grown in 96-well microplates (TPP, Switzerland, Europe) at a
density of 5X10
4
cells, with 100µl of DMEM containing 10% FBS and 10µg/mL
gentamycin. After 24 hours incubation, culture media was discarded and the test
23
product was added (50µL). Incubated cells were exposed to the test products
during 1 (t1), 2 (t2), 3 (t3), 4 (t4) and 5 (t5) minutes. After the exposure time, the
product was aspirated and culture media was added. Cells were incubated at 37°C
and 5% of CO
2
during test times. Cell cultures treated with no product were used
as controls. After incubation for 24 hours (24h), 48 hours (48h) and 7 days (7d),
cells were observed under optical microscopy to evaluate cell morphology.
MTT Assay
Cell viability was evaluated by the MTT assay. It is based on the ability of
the mitochondrial enzyme succinate dehydrogenase to convert the yellow water-
soluble tetrazolium salt 3-[4,5-dimethyl-2-thiazolyl]–2,5–diphenyl-2H-tetrazolium
bromide (MTT) into formazan crystals in metabolically active cells. This water-
insoluble, dark-blue colored product is stored in the cytoplasm of cells, and is
solubilized afterwards, generating a blue color, which is directly proportional to the
amount of metabolically active cells.
After the appropriate incubation period (24h, 48h, 7d), 200µg of MTT
(Sigma, St. Louis, USA) was added to each well of tested cells, followed by 4 hours
incubation at 37°C and 5% CO
2
. Medium was than removed and formazan crystals
were solubilized with 120µL per well of dimethyl sulfoxide (DMSO, Henrifarma, São
Paulo, SP, BR), generating a blue colour. Optical density was read at a wavelength
of 550nm (microplate reader, BIORAD, Japan) [8].
A variety of assays can be used to evaluate cellular viability, each of them
with its own properties. In this study, the MTT test was used due to its simple
24
execution, accessible costs and objective results. The reading of optical density in
the end of the test is proportional to the cellular viability: the bigger the optical
density, the bigger the number of viable cells, and, therefore, smaller toxicity of
tested product [8].
Statistical analysis
Statistical analysis was performed using Statistical Analysis System
statistical (SAS) software, version 6.08. Results were analyzed by one-way
ANOVA and Tukey test. Statistical significance was considered for values of P
<0.05 (95%CI).
25
RESULTS
The analysis of cellular viability for Hep2 cell line, when treated for 1, 2, 3, 4
or 5 minutes with formocresol (FC), tricresol formalin (TC) or formaldehyde (FA) at
different post treatment incubation times (24h, 48h or 7days), showed a reduction
in cell viability in all tested conditions, when compared to the non treated controls
(Table 2). The results observed in different treatment times showed distinct effects
on cell viability (Figure 2). Comparing the effect of different products in different
times of treatment and post treatment incubation on Hep2 cells, a statistically
significant lower toxic effect was observed for FC (p<0.005) (Table 2).
The analysis of FA treatment on Hep2 cells in different times showed a
difference of cytotoxic effect depending on the post treatment incubation time.
When incubation was 24 hours, there was a direct proportional increase in
cytotoxic effect with time exposition; in 48h, an important, but not significant,
difference was observed in the toxic effect (Figure 2).
When NIH3T3 cells were tested, also a reduction in cell viability in all tested
conditions was observed, when compared to the non treated control (Table 3).
A different effect on cell viability was found for the treatment times used
(Figure 3). The analysis of cell treatment with TC on NIH3T3 cells showed a
significant difference in toxicity on cells incubated 24 hours post treatment, with the
lowest toxicity observed at 1 minute time of cells contact with TC. All other
conditions tested showed no significant differences (Figure 3).
The experiments performed with NIH3T3 cells, showed a statistically
significant lower cytotoxic effect for FC in relation to TC and FA, in all tested
26
conditions. The cytotoxic effect of TC was significantly smaller than FA effect only
at 1 minute exposure time (Table 3).
Formaldehyde treatment over NIH3T3 cells showed different results. When
cells incubated 24 hours or 7 days post treatment were analyzed, no significant
difference in cytotoxicity was observed at different treatment times. however, with
cells incubated for 48 hours after treatment, a significant difference was observed
i.e. being 3 minutes the lowest toxic time period and 1 minute the most toxic period
(Figure 3).
Treatment of HeLa cells with test products also resulted in a reduction of cell
viability in all conditions, when compared to the non treated control (Table 4). Cell
viability was differently affected by distinct treatment times (Figure 4).The
experiment with HeLa cells did not show significant difference in cytotoxicity with
FA and TC in the different tested conditions.
However, the treatment with FC resulted in significant difference regarding
toxicity, among treatment times in all post treatment incubation conditions, being 1
minute the most toxic time. The less cytotoxic groups were: for 24 hours incubation
post treatment the 3 minutes time, for 48 hours the 4 minutes time and for 7 days
the 4 and 5 minutes time. The toxic effect observed for FC on HeLa cells was the
lowest among the three test products (Table 4, Figure 4).
27
DISCUSSION
According to Osorio et al, the toxicity of materials used in dental practice is
accessed using a three-step approach. A first step is to screen a candidate
material using a series of in vitro cytotoxicity assays. Then, if the material is
determined not to be cytotoxic in vitro, it can be implanted in subcutaneous tissue
or muscle and the local tissue reaction evaluated. Finally, the in vivo reaction of the
target tissue versus the test material must be evaluated in human subjects or
animals [9].
The results on in vitro cytotoxicity tests of materials used in dentistry may
reflect the effect observed in living tissues [9]. Cell cultures have advantages over
animal experimentation since they afford highly defined culture conditions thereby
avoiding the complex homeostatic mechanisms that occur in vivo [10]. The
European Committee for Standardization is a competent organ for the adoption of
harmonized standards for biological devices, including dental materials. The
committee encourages the increasing study and use of in vitro methods so that
these can be adopted as standards, thereby minimizing the need for in vivo
assessments [11]. Studies using established cell lines and primary cell cultures
concluded that the use of established cellular lines for cytotoxicity tests is
recommended, as they are more resistant to culturing and treatment with cytotoxic
agents [12, 13].
In present study it was evaluated the in vitro cytotoxicity of 3 antiseptical
products used routinely in root canal procedure, FA, TC and FC, in three different
established cell lines (NIH3T3, Hep2 and HeLa) with the MTT cytotoxicity assay.
28
NIH3T3 cells are a fibroblast lineage similar to cells present in dental pulp
and surrounding tissues. Hep2 cells are of epithelial origin from larynx carcinoma
and they regard oral mucosa cells. The HeLa cells come from cervix carcinoma,
and also resemble oral mucosa. Therefore, cell lines used in this experiment are
representative of cells exposed to the process of root canal asepsis. Toxicity profile
of different test products varied among each other and among tested cell lines.
This variation could be explained by the different characteristics of cells, from
biologic origin to ability to grow in vitro.
In the endodontics practice, the FC and TC are used directly into the root
canal, with the aim of asepsis, due to their excellent antimicrobial activity. The TC
is used as a disinfectant in the therapy of root canal with pulp necrosis and
incomplete biomechanical instrumentation. The FC acting as antiseptic and cell
fixative agent is maintained in contact with the pulp of the primary teeth during 5
minutes. However, the formaldehyde present in the formulation of these products
(19%) exerts a toxic effect on cells of the pulp and surrounding tissues [14].
Formaldehyde is a very reactive chemical, and its cytotoxic, mutagenic,
carcinogenic and pro-allergenic potentials are well known and have been of
concern also in dentistry [6, 7].
A clinical study conducted in dogs’ teeth by Garcia-Godoy suggested that
the activity of formocresol during a shorter exposure (one minute) could be
satisfactory and even superior to the so far recommended 5 minutes [15]. In the
present study, TC, FA and FC were tested at 1, 2, 3, 4 and 5 minutes of contact in
vitro with 3 established cell lines, and kept in culture for 24h, 48h and 7 days.
When cell viability was tested, among all products and all conditions, the 1 minute
29
exposure times revealed the lowest number of cells. Considering that cells directly
affected with formaldehyde toxic effect would dye and thus not recover the
proliferation ability, we could speculate that cells surviving after FA treatment could
suffer important alterations, maybe mutations, which would result in a higher
proliferation rate. This could be a reasonable explanation, which remains to be
investigated, once the carcinogenic potential of FA is well known.
The cytotoxic effect observed in all tested conditions for FC on Hep2 cells
was lower than the observed for FA and TC. This data is in accordance with the
proposal of FC formulation, which is based on the reduction of toxicity with the
addition of orto-cresol [16].
In a study conducted by Nilsson et al., the result showed a significantly
higher FA toxicity in fibroblasts than in epithelial cells [17]. In our study, a similar
results regarding cytotoxicity was observed for cells of fibroblast and epithelial
origin. These conflicting results are probably related to the fact that Nilsson et al.
used fibroblast primary culture and HeLa cells in their study, while in our study we
used a fibroblast established cell line (NIH3T3) and HeLa cells.
Cytotoxicity observed for treatment with FC on NIH3T3 cell was the lowest
among test products. Besides, in all incubation times there was a significant
difference between treatment times, with the highest toxicity observed at 1 minute
contact. The presence of orto-cresol in the formulation of TC and FC might have
contributed for this decreased toxic effect, especially in the case of FC (Figure 3).
In our study, all test products were cytotoxic for the different cell lines tested,
in all conditions. Previous studies, investigated toxicity of formaldehyde and cresol
(compounds present in the formulation of FC and TC) in mammal cells, testing cell
30
viability immediately after 24 hours exposure time, and reported a toxic effect of
both products [18, 19]. Besides the different conditions used, their results are in
accordance with ours.
The cytotoxicity of FC on established cell lines was investigated in other
studies which observed a high rate of cell death [20, 21]. In our study, we also
detected a cytotoxic effect of FC, even in the shorter exposure times, but lower
than those observed for TC and FA.
For each cell line and culture time (24h, 48h, 7d) mock controls were used,
consisting of cells submitted to all manipulations, but treated with no product. Cell
viability of mock was considered as reference for products cytotoxicity effect. The
24h and 48h mock cells showed much higher cell viability than treated cells, FC
being the least toxic product. In the 7 days mock cells, a high rate of cell death was
observed, probably due to inhibition contact or lack of nutrients, once incubation
media was not replaced nor subcultures performed during the incubation period.
Our results indicated that all antimicrobial endodontics products evaluated
were toxic in all different tested cell lines, with FC presenting the lowest
cytotoxicity. Therefore, care must be taken when using these chemical agents in
dental practice to minimize their possible adverse effects on human health [18].
The ideal endodontic drug should be bactericidal to all organisms found in the root
canal but must be nontoxic to the periapical tissues. Since the ideal drug has not
been discovered, a drug that most nearly approaches the ideal should be selected
[20]. Reports on in vitro cytotoxicity testing of materials used in endodontics are
relatively poor in the literature, and sometimes may not reflect the in vivo situation.
Therefore, further investigations on endodontics materials are still necessary to find
31
the ideal material that would fulfill all the properties needed for endodontics
materials.
32
REFERENCES
1.Stanford JW. Recommendations for determining biocompatibility and safety for
the clinical use of metals in dentistry. Int Dent J 1986;36:45-8.
2.Menezes MM, Valera MC, Jorge AOC, et al. In vitro evaluation of the
effectiveness of irrigants and intracanal medicaments on microorganisms within
root canals. Int Endod J 2004;37:311-19.
3.Ohara P, Torabinejad M. Kettering JD. Antibacterial effects of various
endodontics medicaments on selected anaerobic bacteria. Journal of Endod
1993;19:498-500.
4.Groth T, Falck P, Miethke RR. Cytotoxicity of biomaterials- basic mechanisms
and in vitro test methods: a review. ATLA 1995:23;790-9.
5.Arenholt-Bindslev D, Bleeg H. Characterization of two types of human oral
fibroblasts with a potential application to cellular toxicity studies: tooth pulp
fibroblasts and buccal mucosa fibroblasts. Int Endod J 1990;23:84-91.
6.Geurtsen W, Leyhausen G. Biological aspects of rot canal filling materials-
histocompatibility, cytotoxicity, and mutagenicity. Clinical oral Investigations
1997;1:5-11.
7.Lewis BB. Chestner SB. Formaldehyde: a review of mutagênic and carcinogenic
potential. Journal of the American Dental Association 1981;103:429-434.
8.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. Journal of Immunological Methods
1983;65:55-63.
9.Osório RM, Hefti A, Vertucci FJ, Shawley AL. Cytotoxicity of endodontic
materials. Journal of Endodontics 1998;24:91-96.
10.Ribeiro DA, Scolastici C, Lima PLA, Marques MEA, Salvadori MF. Genotoxicity
of antimicrobial endodontics compounds by single cell gel (comet) assay in
33
Chinese hamster ovary (CHO) cells. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2005;99:637-40.
11.Schmalz G, Browne RM. The biological evaluation of medical devices used in
dentistry. Intern Dental J 1995;45:275-278.
12.Geurtsen W, Lehmann F, Spahl W, Leyhausen G. Cytotoxicity of 35 dental resin
composite monomers/additives in permanent 3T3 and three human primary
fibroblast cultures. J Biomed Mater Res 1998;41:474-480.
13.Lovschall H, Eiskjaer M, Arenholt-Bindslev D. Formaldehyde cytotoxicity in
three human cell types assessed in three different assays. Toxicology in Vitro
2002;16:63-69.
14.Myers DR, Kenneth H, Dirksen TR, Pashley DH, Whitford GM, Reynolds KE.
Distribution of 14C-formaldehyde after pulpotomy with formocresol. JADA
1978;96:805-813.
15.Garcia-Godoy F. Penetration and pulpal response by two concentrations of
formocresol using two methods of application. J Pedod 1981;5:102-35.
16.‘S-Gravenmade EJ. Some biochemical consideration of fixation in endodontics.
J Endod 1975;1:233-237.
17.Nilsson JA, Zheng X, Sundqvist K, Liu Y, Atzori L, Elfwing A, Arvidson K,
Grafström RC. Toxicity of formaldehyde to human oral fibroblasts and epithelial
cells: influences of culture conditions and role of thiol status. J Dent Res
1998;77:1896-1903.
18.Miyachi T, Tsutsui T. Ability of 13 chemical agents used in dental practice to
induce sister-chromatid exchanges in Syrian hamster embryo cells. Odontology
2005;93:24-29.
19.Hikiba H, Watanabe E, Barrett JC, Tsutsui T. Ability of fourteen chemical agents
used in dental practice to induce chromosome aberrations in Syrian Hamster
Embryo Cells. J Pharmacol 2005;97:146-152.
34
20.Vander Wall GL, Dowson J, Shipman C. Antibacterial efficacy and cytotoxicity
of three endodontic drugs. Oral Surg 1972;33:230-241.
21.Hill SD; Berry CW; Seale NS; Kaga M. Comparison of antimicrobial and
cytotoxic effects of glutaraldehyde and formocresol. Oral Surg Oral Med Oral
Pathol 1991;71:89-95.
35
Table 1
Table 1 Table 1
Table 1 –
––
–
Composition and manufacturers of the tested products
Test products
FC: formocresol TC: tricresol formalin FA: formaldehyde
Manufacturer
Henrifarma, Ibiporã,
PR, BR
Henrifarma, Ibiporã,
PR, BR
Nuclear, Diadema,
SP, BR
Presentation
Liquid Liquid
Liquid
Ingredients
Formaldehyde 19%
orto-cresol 35%
Formaldehyde 19%
orto-cresol 17%
Formaldehyde 38%
solution P.A.
Glycerin Bidistilled water
Absolute ethyl alcohol Absolute ethyl alcohol
Picture 1: Schematical drawings showing to the diverse structures of the tooth (A)
and a apical injury (B)
A
B
enamel
caries
pulp
dentin
Apical lesion
vein
artery
36
Table 2 – Cellular viability of Hep2 cell line incubated for 24 hours, 48 hours and 7
days after treatment with FA, TC and FC
Test product (OD)
24 horas 48 horas 7 dias
Treatment
time
(minutes)
FA
TC
FC
FA
TC
FC
FA
TC
FC
1
B
0,023
B
0,024
A
0,907
B
0,029
B
0,043
A
0,099
B
0,049
B
0,045
A
0,433
2
B
0,020
B
0,036
A
0,812
B
0,030
B
0,049
A
0,091
B
0,043
B
0,045
A
0,522
3
B
0,016
B
0,036
A
0,880
B
0,024
B
0,043
A
0,095
B
0,044
B
0,049
A
0,477
4
B
0,015
B
0,037
A
0,714
B
0,063
B
0,053
A
0,110
B
0,048
B
0,050
A
0,473
5
B
0,016
B
0,093
A
0,942
B
0,062
B
0,051
A
0,108
B
0,051
B
0,062
A
0,507
FC: Formocresol; TC: tricresol formalin; FA: formaldehyde; OD: optical density. OD
of controls: 24h=1.609, 48h=1.109, 7d=0.682; p=0.0001 (Tukey Test). Numbers
represent optical density average of different experiments. Letters (A and B) reefer
to statistical significance of cytotoxic effect observed for all tested products (FA,
TC, FC) in each incubation time (24h, 48h, 7d). Same letter indicates no
statistically significant difference of toxicity between treatment times in each
product; different letters indicate statistically significant difference.
37
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
FA24h FA48h FA7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
0
0,02
0,04
0,06
0,08
0,1
TC24h TC48h TC7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
0
0,2
0,4
0,6
0,8
1
FC24h FC48h FC7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
Figure 2 – Cell viability of Hep2 cell line in different times of treatment (1, 2, 3, 4
and 5 minutes) for each test product: FA (formaldehyde), TC (tricresol formalin)
and FC (formocresol), and incubated for 24 hours, 48 hours and 7 days post
treatment. p=0,999 (Tukey Test). ns: statistically non significant difference.
ns
ns
ns
ns
ns
ns
ns
ns
ns
38
Table 3 - Cellular viability of NIH3T3 cell line incubated for 24 hours, 48 hours and
7 days after treatment with FA, TC and FC
Test product (OD)
24 horas 48 horas 7 dias
Treatment
time
(minutes)
FA
TC
FC
FA
TC
FC
FA
TC
FC
1
C
0,026
B
0,057
A
0,328
B
0,017
B
0,051
A
0,160
B
0,021
B
0,027
A
0,269
2
B
0,022
B
0,052
A
0,515
B
0,018
B
0,042
A
0,360
B
0,019
B
0,030
A
0,381
3
B
0,021
B
0,042
A
0,704
B
0,024
B
0,054
A
0,476
B
0,018
B
0,027
A
0,536
4
B
0,026
B
0,051
A
0,679
B
0,024
B
0,045
A
0,593
B
0,019
B
0,029
A
0,491
5
B
0,050
B
0,050
A
0,588
B
0,023
B
0,055
A
0,602
B
0,028
B
0,025
A
0,464
FC: Formocresol; TC: tricresol formalin; FA: formaldehyde; OD: optical density. OD
of controls: 24h=0.952, 48h=0.660, 7d=0.059; p24h=0.001, p48h=0.0001,
p7d=0.0001 (Tukey Test). Numbers represent optical density average of different
experiments. Letters (A, B and C) reefer to statistical significance of cytotoxic effect
observed for all tested products (FA, TC, FC) in each incubation time (24h, 48h,
7d). Same letter indicates no statistically significant difference of toxicity between
treatment times in each product; different letters indicate statistically significant
difference.
39
0
0,01
0,02
0,03
0,04
0,05
0,06
FA24h FA48h FA7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
0
0,01
0,02
0,03
0,04
0,05
0,06
TC24h TC48h TC7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
FC24h FC48h FC7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
Figure 3 – Cell viability of NIH3T3 cell line in different times of treatment (1, 2, 3, 4
and 5 minutes) for each test product: FA (formaldehyde), TC (tricresol formalin)
and FC (formocresol) and incubated for 24 hours, 48 hours and 7 days post
treatment. pFA24h=0.459; pFA48h=0.050; pFA7d=0.863; pTC24h=0.048;
pTC48h=0.415; pTC7d=0.814; pFC24h=0.043; pFC48h=0.001; pFC7d=0.038
(Tukey Test). ns: statistically non significant difference. Letters (a, b and c)
correspond to statistical significance analysis of cytotoxic effect of tested products
(FA, TC, FC) in each incubation time (24h, 48h, 7d). Same letter indicates no
statistically significant difference of toxicity between treatment times in each
product; different letters indicate statistically significant difference.
ns
b
a ab
ab
ab
ns
ab ab ab
a
b
ns
ns
b
ab
ab
ab
a
a a
ab
b
c
ab
a
ab
a
b
40
Table 4 – Cellular viability of HeLa cell line incubated for 24 hours, 48 hours and 7
days after treatment with FA, TC and FC
Test product (OD)
24 horas 48 horas 7 dias
Treatment
time
(minutes)
FA
TC
FC
FA
TC
FC
FA
TC
FC
1
B
0,019
B
0,008
A
0,156
B
0,018
B
0,036
A
0,306
B
0,044
B
0,049
A
0,392
2
B
0,026
B
0,006
A
0,234
B
0,017
B
0,044
A
0,390
B
0,038
B
0,048
A
0,501
3
B
0,018
B
0,009
A
0,591
B
0,013
B
0,035
A
0,453
B
0,057
B
0,044
A
0,459
4
B
0,019
B
0,008
A
0,477
B
0,015
B
0,038
A
0,517
B
0,041
B
0,053
A
0,579
5
B
0,021
B
0,005
A
0,553
B
0,012
B
0,035
A
0,347
B
0,038
B
0,054
A
0,621
FC: Formocresol; TC: tricresol formalin; FA: formaldehyde; OD: optical density. OD
of controls: 24h=0.922, 48h=0.738, 7d=0.070; p24h=0.001, p48h=0.0001,
p7d=0.0001 (Tukey Test). Numbers represent optical density average of different
experiments. Letters (A and B) reefer to statistical significance of cytotoxic effect
observed for all tested products (FA, TC, FC) in each incubation time (24h, 48h,
7d). Same letter indicates no statistically significant difference of toxicity between
treatment times in each product; different letters indicate statistically significant
difference.
41
0
0,01
0,02
0,03
0,04
0,05
0,06
FA24h FA48h FA7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
0
0,01
0,02
0,03
0,04
0,05
0,06
TC24h TC48h TC7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
FC24h FC48h FC7d
Incubation post treatment
Optical Density (OD)
t1
t2
t3
t4
t5
Figure 4 – Cell viability of HeLa cell line in different times of treatment (1, 2, 3, 4
and 5 minutes) for each test product: FA (formaldehyde), TC (tricresol formalin)
and FC (formocresol) and incubated for 24 hours, 48 hours and 7 days post
treatment. pFA24h=0.191; pFA48h=0.125; pFA7d=0.406; pTC24h=0.144;
pTC48h=0.823; pTC7d=0.174; pFC24h=0.043; pFC48h=0.0042; pFC7d=0.033
(Tukey Test). ns: statistically non significant difference. Letters (a, b and c)
correspond to statistical significance analysis of cytotoxic effect of tested products
(FA, TC, FC) in each incubation time (24h, 48h, 7d). Same letter indicates no
statistically significant difference of toxicity between treatment times in each
product; different letters indicate statistically significant difference.
bc
bc
a
c
ab
b
ab
a
ab
a
ab
ab
a
b
ab
ns
ns
ns
ns
ns
ns
42
CONSIDERAÇÕES FINAIS
A proposta deste estudo foi avaliar a toxicidade de produtos usados na
endodontia para desinfecção do canal radicular: formocresol, tricresol formalina e
formaldeído, em nível celular, através de testes de citotoxicidade em linhagens
celulares estabelecidas. A partir dos resultados obtidos, é possível concluir que:
• O formocresol, o tricresol formalina e o formaldeído apresentaram
citotoxicidade quando testados na linhagem celular Hep2 (células epiteliais de
carcinoma de laringe humano)
• O formocresol, o tricresol formalina e o formaldeído apresentaram
citotoxicidade quando testados na linhagem celular NIH3T3 (fibroblastos de
camundongos)
• O formocresol, o tricresol formalina e o formaldeído apresentaram
citotoxicidade quando testados na linhagem celular HeLa (células originárias de
câncer cervical humano)
• O formocresol, o tricresol formalina e o formaldeído foram citotóxicos nos
diferentes tempos de exposição testados (1, 2, 3, 4, e 5 minutos), nas três
linhagens celulares (Hep2, NIH3T3, HeLa)
• O efeito citotóxico do formocresol, do tricresol formalina e do formaldeído
permaneceu após 24 horas, 48 horas e 7 dias de incubação pós-tratamento,
nas três linhagens celulares testadas (Hep2, NIH3T3, HeLa)
43
• O formocresol foi o produto que apresentou menor citotoxicidade, em todas as
condições testadas, sendo a diferença de toxicidade estatisticamente
significante quando comparada com o tricresol formalina e o formaldeído.
44
ANEXOS
45
Hep 2
NIH3T3
HeLa
TC TCTCFC FC FCFA FA FA
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
1 min
24h
48h
7d
2 min
24h
48h
7d
3 min
24h
48h
7d
4 min
24h
48h
7d
5 min
24h
48h
7d
46
Anexo 2: Especificações da linhagem celular HeLa utilizada (Fonte: ATCC)
HeLa (human, Black, cervix, carcinoma, epitheloid)
IZSBS BS TCL20
Morphology: epithelial-like
Species: human, Black female 31 years old; Tissue: cervix; Tumor: carcinoma,
epitheloid
Properties: antitumour testing; transformation; tumorigenicity; cytotoxicity; cell
biology; bacterial invasiveness; virology; Susceptible to: adenovirus 3, measles,
poliovirus 1, echovirus, vaccinia, arbovirus, respiratory syncytial virus, reovirus 3,
rhinovirus, Coxsackie
Available in the following LABORATORY:
• Istituto Zooprofilattico Sperimentale (IZSBS, Brescia)
continuous culture, grown as monolayer; MEM (EBSS) + 10% FBS; 37C,
5% CO2
Further information
Freezing medium: Culture medium + 10% Glycerol; mycoplasma negative,
culture
Karyology: aneuploid
tumorigenic in nude mice
Availability in cell line catalogues: ATCC CCL 2; ECACC 85060701; DSMZ ACC
57; ICLC HTL95023;
47
Anexo 3: Especificações da linhagem celular Hep2 utilizada (Fonte: ATCC)
Hep 2 (human, Caucasian, larynx, carcinoma, epidermoid)
IZSBS BS TCL 23
Morphology: epithelial-like
Species: human, Caucasian male 56 years old; Tissue: larynx; Tumor: carcinoma,
epidermoid; Validated by isoenzymes: confirmed as human with NP, G6PD, PEPB,
AST, LD
Depositor: obtained from Centro Virus Respiratori, Roma (I)
Properties: virology; expressing xenobiotic metabolising enzymes; Susceptible to:
adenovirus 3, poliovirus 1, herpes simplex, vesic. stomatitis (Indiana), respiratory
syncytial virus
Available in the following LABORATORY:
• Istituto Zooprofilattico Sperimentale (IZSBS, Brescia)
continuous culture, grown as monolayer; MEM (EBSS) + 10% FBS; 37C,
5% CO2 Passages: 363
Further information
Freezing medium: Culture medium + 10% DMSO; mycoplasma negative,
culture
Karyology: 2n = 46 in 47 cells
tumorigenic
Availability in cell line catalogues: ATCC CCL 23; ECACC 86030501;
Bibliographic references:
• Cancer Res 1955;15:598
• J Clin Microbiol 1990;28:1049 - PMID: 2161864
• J Gen Virol 1983;64:825 - PMID: 6834007
• Virology 1969;38:42 - PMID: 4306525
48
Anexo 4: Especificações da linhagem celular NIH3T3 utilizada (Fonte: ATCC)
NIH 3T3 (mouse, NIH Swiss, embryo)
ECACC 93061524
Morphology: Fibroblast
Mouse Swiss NIH embryo contact inhibited
Depositor: Obtained from ATCC, USA
No restrictions. Patent: None Specified By Depositor
Properties: Applications: DNA transfection studies
Available in the following LABORATORY:
• CAMR Centre for Applied Microbiology & Research (ECACC, Salisbury,
Wiltshire)
DMEM + 2mM Glutamine + 10% Calf Serum (CS). Split confluent cultures
1:2 to 1:6 i.e. seeding at 2-5x10,000 cells/cm2 using 0.25% trypsin/EDTA;
5% CO2; 37C. Do not allow culture to become fully confluent; the use of
fetal calf serum is not recommended. Passages: 136
Hazard: CY
Established from a NIH Swiss mouse embryo. These cells are highly contact
inhibited and are sensitive to sarcoma virus focus formation and leukaemia
virus propagation.
Availability in cell line catalogues: ATCC CRL 1658; DSMZ ACC 59;
Bibliographic references:
J Virol 1969;4:549; Cell 1979;16:63; Cell 1979;16:347
49
Anexo 5: Produtos utilizados nos testes de viabilidade celular
Formaldeído (FA)
Formocresol (FC)
Tricresol formalina (TC)
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