FACULDADE DE ODONTOLOGIA DE PIRACICABA
THAYSE RODRIGUES DE SOUZA
RELAÇÃO ENTRE A ATIVIDADE DA ANIDRASE CARBÔNICA VI,
ALFA-AMILASE SALIVAR, CAPACIDADE TAMPÃO, FLUXO
SALIVAR E CÁRIE DENTAL EM CRIANÇAS
RELATIONSHIP AMONG SALIVARY CARBONIC ANHYDRASE VI
ACTIVITY, ALPHA-SALIVARY AMYLASE, BUFFERING CAPACITY,
SALIVARY FLOW RATE AND DENTAL CARIES IN CHILDREN
Piracicaba 2016
RELAÇÃO ENTRE A ATIVIDADE DA ANIDRASE CARBÔNICA VI,
ALFA-AMILASE SALIVAR, CAPACIDADE TAMPÃO, FLUXO
SALIVAR E CÁRIE DENTAL EM CRIANÇAS
RELATIONSHIP AMONG SALIVARY CARBONIC ANHYDRASE VI
ACTIVITY, ALPHA-SALIVARY AMYLASE, BUFFERING CAPACITY,
SALIVARY FLOW RATE AND DENTAL CARIES IN CHILDREN
Piracicaba 2016
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Odontologia, na Área de Odontopediatria.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dentistry in Pediatric Dentistry Area.
Orientador: Profa. Dra. Marinês Nobre dos Santos Uchôa
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA THAYSE RODRIGUES DE
SOUZA E ORIENTADA PELA PROFa. DRa MARINÊS
À Deus por me guiar nos diversos caminhos que se abriram para mim... Por me iluminar nas decisões mais difíceis... Por ser minha fortaleza, refúgio e morada espiritual e por sempre estar comigo em qualquer lugar que eu vá.
Aos meus pais Ana Dalva e Antônio Rodrigues por terem me dado a oportunidade de estudar e sempre me guiarem a este caminho... Por sempre me apoiarem em minhas decisões e pelo carinho e amor sustentadores. Obrigada não só por me dar a vida, mas principalmente por me ensinar a vivê-la.
Ao meu querido esposo Jorge Leão pelo amor, carinho, incentivo e companheirismo fundamental... Por sua mão sempre estendida a me ajudar, por ser parte de mim, parte de quem eu sou e por tornar meus dias imensamente felizes.
À minha orientadora, Profa. Dra. Marinês Nobre dos Santos Uchôa, por ter me aceitado como sua orientanda, pela edificante orientação, por me proporcionar mais uma experiência da pesquisa científica e sempre acreditar em minha dedicação e empenho, por todos os ensinamentos e compreensão quando mais precisei.
Às crianças que fizeram parte dessa pesquisa... Meus amores, vocês foram fundamentais e me deram incentivo a cada dia... Obrigada pelo olhar de pureza, felicidade e carinho que me passavam em cada dia de coleta.
À Força Aérea Brasileira nas pessoas do Coronel Médico Laerte Lobato de Moraes, diretor do Hospital de Aeronáutica de Belém e Tenente-Coronel Luiz Fernando da Costa
Tavares, chefe da Divisão Odontológica, pela compreensão e apoio no seguimento de meu
curso de doutorado.
À minha irmã Thalyta Souza e às famílias Souza, Rodrigues e Leão pelo enorme carinho apoio e por sempre torcerem pelas minhas conquistas.
À Universidade Estadual de Campinas, na pessoa do reitor Prof. Dr. José Tadeu
Jorge, à Faculdade de Odontologia de Piracicaba FOP-UNICAMP, na pessoa do seu diretor
Prof. Dr. Guilherme Elias Pessanha Henriques, à Comissão de Pós-Graduação da FOP-UNICAMP na pessoa da presidente Profa. Dra. Cínthia Pereira Machado Tabchoury e da Coordenadora do Programa de Pós-Graduação em Odontologia Profa. Dra. Juliana Trindade Clemente Napimoga, pela participação dessa conceituada instituição no meu crescimento científico.
À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo apoio financeiro concedido durante o desenvolvimento dessa tese.
Às Profas. Dras., Fernanda Miori Pascon, Maria Beatriz Duarte Gavião, Regina
Maria Puppin Rontani do Departamento de Odontologia infantil, por terem me recebido tão
bem quando cheguei à faculdade, por todos os conhecimentos passados, tanto os conteúdos relacionados à Odontopediatria quanto àqueles relacionados ao ensino e à pesquisa.
A todos os professores do Programa de Pós-Graduação em Odontologia da FOP-UNICAMP e aqueles professores convidados a ministrar diversas aulas que engrandeceram nossos conhecimentos científicos principalmente no campo do ensino e pesquisa.
Aos professores colaboradores, Prof. Dr. Sérgio Line e Prof. Dr. Marcelo Marques pelo desenvolvimento do protocolo de visualização da atividade da enzima anidrase carbônica VI que possibilitou a execução desse projeto de tese.
Ao técnico do laboratório de Odontopediatria, Marcelo Corrêa Maistro, pelo auxílio fundamental nas etapas laboratoriais da pesquisa.
À Secretaria Municipal de Educação do município de Piracicaba por ter permitido a realização da pesquisa.
Às diretoras das creches visitadas por terem me acolhido tão bem na ocasião da coleta de dados e amostras da pesquisa.
À Professora Dra. Cínthia Pereira Machado Tabchoury e Prof. Dra. Maria Beatriz Duarte Gavião membros da banca de pré-qualificação pelas sugestões para execução desse trabalho.
Ao Prof. Dr. Natanael Barbosa e Prof. Dr. Milton Duarte, do Departamento de Cariologia da Universidade Federal de Alagoas- Faculdade de Odontologia pelo exemplo de pesquisadores, por terem me iniciado na pesquisa científica na graduação em Odontologia, tendo sido fundamentais na escolha dos caminhos no ensino e pesquisa. Ao Prof. Dr. Luiz
Alcino Monteiro Gueiros e Prof. Dr. Jair Carneiro Leão do Departamento de
Estomatologia da Universidade Federal de Pernambuco- Faculdade de Odontologia, meus orientadores do mestrado pelos conhecimentos passados e que foram fundamentais para a subida de mais um degrau em minha formação acadêmica.
À Maria Elisa dos Santos, Eliane Melo Franco de Souza, Érica A. Pinho Sinhoreti e Raquel Q. M. Cesar Sacchi e Roberta C. Morales dos Santos, pela ajuda e atenção nas etapas administrativas e a todos os funcionários da FOP-UNICAMP, pela colaboração.
Às amigas Lívia Pagotto e Fabiana Furtado por terem me acolhido tão bem na cidade na ocasião de minha chegada pela amizade e companhia diária.
Às amigas Lívia Pagotto e Bruna Raquel por o auxílio em etapas da pesquisa.
À amiga Andréia Alves, pela mão estendida não só para aprender, mas também ajudar. Obrigada pela companhia nas muitas horas esperando as bandas da anidrase e pela amizade.
Aos colegas e amigos: Maria Carolina S. Marquezin, Marina S. Leme, Bárbara
Lucas, Ana Bheatriz M. Montes, Filipe Martins, Alexsandra S. Iwamoto, Ariany B. Carvalho, Bruna R. Zancopé, Lívia P. Rodrigues, Luciana T. Inagaki, Vanessa Benetello, Micaella Cardoso, Natalia Martins, Darlle Araújo, Thais Varanda e Lenita Lopes pelo convívio e amizade durante essa importante etapa.
Aos amigos da Força Aérea Brasileira em especial às Tenentes Patrocínio, Paola, Flávia Carvalho, Camilla Pinto, Camila Rocha, Kobayashi, Luciane Bertoldi, Cibelle, Glauce Vaz , Thayanna e Valéria pela amizade e carinho.
“Bom mesmo é ir a luta com determinação, abraçar a vida com paixão, perder com classe e vencer com ousadia, porque o mundo pertence a quem se atreve (e tem fé) e a vida é muito para ser insignificante.” Augusto Branco
VI é responsável por catalisar a principal reação tamponante da cavidade bucal. A enzima α-amilase é responsável pela formação da película, biofilme e no metabolismo do amido. Não há relatos na literatura que tenham investigado longitudinalmente a relação entre a AC VI e cárie dental ou transversalmente a atividade de α-amilase logo após um desafio cariogênico. A tese foi apresentada em dois Capítulos. Os objetivos do Capítulo 1 foram: Determinar o fluxo salivar estimulado (FSE), capacidade tampão (CT) e a atividade de AC VI na saliva de crianças com cárie e livres de cáries antes e após o bochecho com solução de sacarose a 20% e investigar a relação entre essas variáveis e a cárie dental longitudinalmente após um ano e no Capítulo 2: Investigar a atividade de α-amilase na saliva de crianças com cárie e livres de cáries antes e após o bochecho com uma solução de sacarose a 20% e sua relação com FSE, CT e a cárie dental transversalmente. No Capítulo 1 foram alocadas 47 crianças de 48 a 78 meses de idade, divididos em três grupos após cálculo do incremento de cárie após um ano: grupo livre de cárie (LC, n=10), grupo com cárie (C, n=20) e grupo de cárie paralisada (CP, n=17). No Capítulo 2, 38 crianças de 48 a 77 meses de idade, divididas em dois grupos: com cárie (C, n=20) e livres de cárie (LC, n=18). A atividade da AC VI foi quantificada por zimografia. O FSE foi expresso em mL/min. A CT foi medida pelo método de Ericsson por meio de um eletrodo de pH conectado a um peagâmetro. A análise de α-amilase foi realizada por ensaio enzimático. Os dados de AC VI foram submetidos ao teste de Wilcoxon e Kruskall-Wallis para comparações pareadas dos valores antes e depois do bochecho e comparação entre grupos respectivamente. Os dados de FSE e CT foram submetidos aos testes acima mencionados nos dois Capítulos. Os dados da atividade de α-amilase foram submetidos aos testes T de Student pareado e independente. Foi realizado também análise de correlação de Spearman (α=0.05). Os resultados do Capítulo 1 mostraram que a atividade de AC VI apresentou um decréscimo significativo após o bochecho nos grupos LC no baseline e após um ano e no grupo CP somente após um ano (p= 0.037, p=0.028 e p=0.027, respectivamente). Não se observou mudanças na atividade de AC VI no grupo CL antes e depois do bochecho nos dois períodos do estudo. A atividade de AC VI antes do bochecho no baseline exibiu correlação negativa significativa como índice de cárie no baseline antes e depois do bochecho e após um ano antes do bochecho no grupo C (0.609, p=0.004 e r=-0.516, p=0,020, r= -0.545, p=0.013, respectivamente). Uma correlação negativa significativa foi encontrada entre o índice de cárie nos dois tempos do estudo e CT após o bochecho após
bochecho diferindo significativamente do grupo LC (p=0.024 e p=0.019). Observou-se nos dois Capítulos aumento do FSE após o bochecho e diminuição dos valores de CT após o bochecho com sacarose. Conclui-se que a atividade de AC VI exerce possível participação no controle de pH bucal após um desafio cariogênico, principalmente em crianças com cárie. Sugere-se ainda, uma possível participação da α-amilase como facilitadora do processo de cárie devido ao aumento de sua atividade quando as crianças com cárie foram submetidas a um desafio cariogênico.
Palavras-chave: Anidrase carbônica VI, fluxo salivar, capacidade tampão da saliva, cárie
saliva. AC VI is responsible for catalyzing the main reaction buffering the oral cavity. SAA is associated with the pellicle and biofilms formation and starch metabolism. There are no reports in the literature that have longitudinally investigated the relationship between AC VI and dental caries and a cross-sectional study to investigate the SAA activity after a cariogenic challenge. The objectives of the Chapter 1 of this thesis were: Determine the stimulated salivary flow (SSFR), buffer capacity (BC) and CA VI activity in the saliva of children with caries and caries-free before and after rinsing with a sucrose solution to 20% and to investigate the relationship of these variables with dental caries in a longitudinal study of one year of follow-up. And of the Chapter 2: Investigate the SAA activity in saliva of children with caries and caries-free before and after rinsing with a sucrose solution at 20% and its relationship with SSFR, BC and dental caries in a cross-sectional study. Were allocated to the study of Chapter 1 47 children 48-78 months age, divided into three groups after calculation of caries increment after one year: caries free group (CF), caries lesion group (CL) and arrestment caries group (AC). And in Chapter 2, 38 children aging 48-77 months old, divided into two groups: caries lesion group (CL) and caries free group (CF). The activity of CA VI was quantified by zymography. The SSFR was expressed in mL/min. The BC was measured by Ericsson’s method. The SAA activity was analyzed by the enzyme kinetic assay. Wilcoxon test and the Kruskal-Wallis test for paired comparisons of the values of CAVI before and after the rinses and comparison between groups respectively. To SSFR and BC data were employed the tests mentioned above in the two Chapters. The Student t test paired and independent were employed to the SAA data. It was also performed Spearman correlation analysis (α = 0.05). The results of chapter 1 show that CA VI activity significantly decreased after the cariogenic challenge at the CF group in baseline and follow-up and at AC group only at the follow-up (p= 0.037, p=0.028 e p=0.027, respectively). No change in CA VI activity was found at the two periods of the study in CL group. Salivary CA VI activity before rinse at the baseline shows also a negative correlation with dental caries at the baseline before and after rinse and at the follow-up before the rinse in the CL group (0.609, p=0.004 e r=-0.516, p=0,020, r= -0.545, p=0.013, respectively). A negative correlation was found between dental caries at baseline as well at follow-up and BC after rinse at follow-up (r=-0.345, p=0.017 e r=-0.303, p=0.038 respectively). The results of Chapter 2 shows that the CL group exhibited a significant increase on SAA activity after rinse (p=0.001), and significantly
concluded that the AC VI activity possible participates on the oral pH control after a cariogenic challenge, particularly in children with caries. It is also suggested possible involvement of SAA as a facilitator of the decay process due to the increase of its activity when the children were submitted to a cariogenic challenge in the group of children with caries.
KEY-WORDS:Carbonic anhydrase VI, salivary flow, salivary buffer capacity, early childhood
FIGURA 1. Exame clínico para avaliação do índice de cárie em pré-escolares do município de Piracicaba-SP (Capítulo 1 e 2)
91
FIGURA 2.Cárie precoce da infância (Capítulo 1 e 2) 92
FIGURA 3.Forma de coleta de saliva estimulada (capítulos 1 e 2) 93 FIGURA 4.Bochecho com solução de sacarose 20% (Capítulos 1 e 2) 94
FIGURA 5.Material utilizado na coleta de saliva (Capítulos 1 e 2) 95 FIGURA 6.Metodologia de avaliação da capacidade tampão (Capítulos 1 e 2) 95
FIGURA 7. Metodologia de avaliação da atividade do fluxo salivar estimulado (Capítulos 1 e 2)
96
FIGURA 8. Metodologia de avaliação da atividade da enzima Anidrase Carbônica VI (Capítulo 1)
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FIGURA 9. Metodologia de avaliação da atividade da enzima α-amilase salivar
(Capítulo 2)
CPI Cárie precoce da infância
AC VI Anidrase carbônica VI
AC II Anidrase carbônica II
CPOD Índice de cariados, perdidos e obturados
FSE Fluxo salivar estimulado
CT Capacidade tampão
CA VI Carbonic anhydrase VI
SSFR Stimulated salivary flow rate
CF Caries free group
CL Caries lesions group
AC Arrested caries group
CO2 Gás carbônico
HCO3- Íon Bicarbonato
WHO+ECL World Health Organization diagnostic
criteria and the early caries lesions
IQR Interquatile range
dmfs+ ECL Decayed, missing and filled surfaces plus
early caries lesions
CA II Carbonic anhydrase II
AC II Anidrase Carbônica II
SAA Salivary α-amylase
1. INTRODUÇÃO 17
2. ARTIGOS 23
2.1 Artigo: Relationship among dental caries and salivary carbonic anhydrase VI activity, buffer capacity and flow rate – A longitudinal study in children
23
2.2 Artigo: Sucrose increases salivary α-amylase activity in saliva of children- A cross-sectional study
46
3. DISCUSSÃO 69
4. CONCLUSÃO 77
REFERÊNCIAS 78
APÊNDICE – Produção bibliográfica da aluna 85
ANEXOS 86
Anexo 1 - Certificado do Comitê de Ética em Pesquisa da FOP- UNICAMP 86 Anexo 2 - Autorização da Secretaria Municipal de Saúde de Piracicaba-SP para
realização da pesquisa
87
Anexo 3 - Ficha clínica utilizada na coleta de dados 88
Anexo 4 - Declaração 89
1 INTRODUÇÃO
A cárie precoce da infância (CPI), uma apresentação agressiva da cárie dental, tem início com lesões de manchas brancas nas faces vestibulares de incisivos decíduos superiores ao longo da margem gengival (AAPD, 2008). A doença em crianças é associada a fatores como, hábitos alimentares inapropriados, alto consumo de carboidratos, medidas de higiene bucal deficientes e baixo poder socioeconômico (Parisotto et al., 2010). Se não tratada, a doença pode destruir a dentição decídua, causar dor e desconforto, infecção aguda, insuficiências nutricionais, problemas de fala e aprendizagem (AAPD, 2008, Parisotto et al., 2010).
A prevalência da CPI é alta e sua severidade aumenta com a idade. Além disso, uma pesquisa longitudinal recentemente realizada demonstrou que pré-escolares com CPI apresentaram risco 17 e 24 vezes maiores de desenvolverem novas lesões de manchas brancas ativas e de apresentarem lesões de cárie cavitadas, respectivamente (Parisotto et al., 2012). Levantamentos epidemiológicos evidenciaram também, que no Brasil a doença apresenta-se como um problema de saúde pública (Ferreira et al., 2007, Moimaz et al., 2016). No último relatório de saúde bucal, Projeto SB Brasil 2010 (Ministério da Saúde), apenas 46,6% das crianças brasileiras aos cinco anos de idade apresentou-se livre de cárie na dentição decídua e 43,5% aos 12 anos, já na dentição permanente (Ministério da Saúde, 2010).
A cárie dental é uma doença biofilme-sacarose dependente resultado do desequilíbrio do biofilme no meio ambiente bucal o que contribue assim para a agregação e metabolismo bacteriano na superfície dos dentes (Marsh, 2009, Sheiham e James, 2015). Neste aspecto, a saliva é um fator de proteção fundamental que participa do processo de cárie tanto na dentição decídua quanto na permanente (Laine et al., 2014). A saliva tem em sua composição vários mecanismos de defesa, que incluem imunoglobulinas (IgA, IgG e IgM), proteínas aglutinantes e várias enzimas (lactoferrina, lisozima, e peroxidades) oriundas do plasma e de células acinares, que interferem no crescimento microbiano (Kivela et al., 1999a, Gao et al., 2016).
Não apenas a composição da saliva, mas também fatores como o fluxo salivar e a capacidade tampão são extremamente importantes na dinâmica do processo de cárie (Cunha-Cruz et al., 2013). O fluxo salivar é o parâmetro salivar mais importante neste processo, pois a atividade cariostática ou eficácia de praticamente todos os outros parâmetros salivares (capacidade tampão salivar, agentes antimicrobianos) dependem do fluxo salivar (Lagerlof e Oliveby, 1994, Tenovuo, 1997, Laine et al., 2014).
O fluxo salivar normal é um fator altamente protetor contra a cárie, uma vez que geralmente está associado ao pH e à capacidade tampão salivar elevados, pois provoca um aumento de todos os componentes salivares. Por outro lado, há uma correlação mais fraca entre uma baixa capacidade tampão da saliva e o aumento do índice de cárie (Leone e Oppenheim, 2001). No entanto, já foi demonstrada uma clara relação inversa entre a capacidade tampão salivar e suscetibilidade à cárie (Ericsson, 1959). Estudos previamente realizados mostraram que crianças com cárie apresentavam baixos valores de capacidade tampão (Bhayat et al., 2013, Kuriakose et al., 2013). No entanto, a presença de baixos valores de capacidade tampão ainda não é considerada fator de risco para a ocorrência da doença cárie (Gao et al., 2016).
Para evitar que o pH diminua a um nível crítico, a saliva contém mecanismos tamponantes específicos (Llena-Puy, 2006). A capacidade tampão da saliva envolve três sistemas tamponantes que são o bicarbonato, o fosfato e as proteínas salivares, de forma que esses três sistemas trabalham em diferentes intervalos de pH. Enquanto que a atividade tampão ótima dos sistemas bicarbonato e fosfato ocorre em valores de pKa 6.1-6.3 e 6.8-7.2, respectivamente, o sistema de proteínas salivares atua de forma efetiva em valores de pKa em torno de 4,0 (Bardow et al., 2000, Cheaib et al., 2012). No entanto, a concentração destas macromoléculas na saliva é baixa, e em condições normais, estas, não são muito importantes como substâncias tampão na saliva (Fejerskov e Kidd, 2007).
O sistema tampão mais importante em condições de estimulação salivar é o sistema bicarbonato, que é responsável por 70 a 90% da capacidade tampão da saliva total. Baseia-se no equilíbrio do ↑CO2 + H2O ↔ H2CO3↔ HCO3- + H+ onde a concentração de bicarbonato tende a aumentar com a estimulação do fluxo salivar (Lilienthal, 1955, Izutsu, 1981, Bardow et al., 2000). Uma característica importante e exclusiva deste sistema é a conversão do gás carbônico do estado dissolvido para o estado volátil. Quando o ácido é adicionado essa conversão de estados aumenta a eficácia da neutralização, não havendo acúmulo de produtos finais, mas a completa remoção de ácido, o que é conhecido como “fase tampão”(Kivela et al., 1999a). Esta reação na cavidade oral e no trato alimentar alto é catalisada pela enzima anidrase carbônica VI (AC VI) que está presente na saliva (Kivela et al., 1999a, Kimoto et al., 2006).
As anidrases carbônicas são metaloenzimas de zinco que participam da manutenção da homeostase do pH em vários tecidos e fluidos biológicos do corpo humano catalisando a
reação de hidratação reversível do dióxido de carbono, CO2 + H2O ↔ HCO3- + H+ (Sly e Hu, 1995, Pastorekova et al., 2004). Dentre as 16 isoenzimas isoladas de mamíferos, pelo menos duas (AC II e AC VI) estão envolvidas na fisiologia salivar, uma vez que expressas nas glândulas salivares de humanos, participam da regulação do pH no meio bucal (AC VI) e da secreção de bicarbonato na saliva (AC II) (Kadoya et al., 1987, Parkkila et al., 1990, Supuran e Scozzafava, 2007). Em humanos, a AC VI é produzida unicamente pelas células acinares serosas das glândulas parótidas e submandibulares e é secretada na saliva, seguindo o ritmo circadiano, com baixa concentração durante o sono, aumentando rapidamente ao acordar e após a primeira refeição (Parkkila et al., 1990, Parkkila et al., 1995). Tal secreção é muito semelhante à da enzima α-amilase salivar, e uma correlação positiva foi encontrada entre o nível de atividade de α-amilase salivar e a concentração de AC VI, sugerindo-se que as duas enzimas poderiam ser secretadas pelos mesmos grânulos e mecanismos secretórios (Parkkila et al., 1995, Kivela et al., 1999b).
O papel fisiológico da AC VI salivar tem sido esclarecido nos últimos anos (Leinonen et al., 1999, Kivela et al., 1999b, Kivela et al., 2003, Kimoto et al., 2006, Frasseto et al., 2012). Pesquisas previamente realizadas demonstraram que a AC VI salivar pode ser considerada uma proteína anti-cárie na saliva (Leinonen et al., 1999, Kimoto et al., 2006). Quando da exposição do biofilme à sacarose, ocorre uma queda do pH no intervalo de poucos minutos, o que pode levar à dissolução do mineral do esmalte. Esse femômeno continua ocorrendo até que o pH retorne ao valor acima do pH crítico do esmalte (Dawes, 2008). O mecanismo pelo qual esta isoenzina atua no controle do pH, sugere que a AC VI liga-se a película de esmalte e facilita a neutralização ácida pelo bicarbonato salivar (Leinonen et al., 1999). No biofilme dental, a AC VI fica situada em sítios ideais para catalisar a reação reversível de conversão de bicarbonato salivar e íons de hidrogênio fornecidos por bacterias cariogênicas, em dióxido de carbono e água (HCO3 + H+ ↔ CO2 + H2O) (Leinonen et al., 1999, Kimoto et al., 2006). O estudo de Kimoto et al. (2006) evidenciou a presença desta enzima no biofilme dental, sendo mostrado uma diminuição do pH do biofilme, quando a cavidade bucal era submetida a um bochecho com solução de acetazolamida, inibidor específico da enzima AC VI. Esses autores sugerem que pelo mecanismo catalisador exercido pela enzima, a AC VI seja capaz de prover uma maior neutralização dos ácidos do biofilme dental.
A literatura aponta que ao catalisar o sistema tampão mais importante da cavidade bucal, o mecanismo de ação de AC VI protege a superfície dental pela neutralização dos ácidos nesse micro ambiente. Pesquisas nessa área tem indicado ainda resultados inconclusivos. Algumas têm indicado uma correlação negativa entre a concentração salivar AC VI e a experiência de cárie (Szabo, 1974, Kivela et al., 1999b). O estudo realizado por Szabó (1974) mostrou que a saliva de crianças de 7 a 14 anos de idade e livres de cárie, expressava uma maior concentração da AC VI do que aquela de crianças com cárie. Posteriormente, Kivela et al. (1999b) mostraram também que baixas concentrações de AC VI na saliva pareciam estar associadas a um aumento na prevalência de cárie, particularmente em adultos jovens com a higiene bucal negligenciada. Por outro lado, ao investigar a atividade da AC VI antes e após um bochecho de sacarose a 20%, Frasseto et al. (2012), observaram que a variação da atividade da isoenzima foi significativamente maior na saliva de pré-escolares com cárie quando comparada aqueles livres de cárie. Esses autores observaram também uma correlação negativa entre a variação da atividade da isoenzima e o índice de cárie. Encontraram ainda, maior atividade da enzima antes do bochecho no grupo com cárie (p=0.051). Os resultados de Ozturk et al. (2008) e Yarat et al. (2011), não mostraram diferença significativa na concentração de AC VI entre grupos com e sem cárie, no entanto, Ozturk et al. (2008) encontraram uma correlação negativa significativa entre a concentração de proteínas total e o índice CPOD de adultos jovens, sugerindo a diminuição na concentração de proteínas protetoras na saliva de indivíduos com cárie.
Resultados contraditórios também são encontrados na literatura. Culp et al. (2013), encontraram marcada contribuição da deleção do gene que transcreve a AC VI na redução de cáries em ratos. Por outro lado Li et al. (2015) encontraram a presença significativa do genótipo polimórfico do gene rs17032907, transcritor de AC VI em indivíduos com susceptibilidade à cárie. Ainda, a análise da literatura relacionada à AC VI evidencia que, com exceção da pesquisa realizada por Frasseto et al. (2012) e Aidar et al. (2013) que analisaram a atividade de AC VI, todas determinaram apenas a concentração da AC VI na saliva ou biofilme. No entanto, uma alta concentração de AC VI na saliva ou biofilme não necessariamente significa que toda isoenzima presente nestes meios esteja ativa e assim, possa exercer o seu efeito. Além disso, não se tem conhecimento de pesquisas longitudinais que tenham investigado a atividade da AC VI no início e na progressão da cárie dentária em crianças. Dessa forma, a determinação da atividade de AC VI na saliva pode fornecer
evidências adicionais dos efeitos desta isoenzima na dinâmica do processo de cárie no que concerne o seu início e progressão.
Um dos componentes mais abundantes da saliva é a enzima α-amilase, que é produzida e secretada pelas células epiteliais acinares das glândulas salivares, principalmente as glândulas parótidas. A enzima exerce na saliva atividade hidrolítica, responsável pela quebra inicial de amido em carboidratos de baixo peso molecular, que são substratos fermentados por várias espécies de bactérias presentes na cavidade bucal (Rogers et al., 2001). Linhas de evidência apontam para a participação da α-amilase na formação do biofilme dental, uma vez que esta enzima é um constituinte abundante da película adquirida (Scannapieco et al., 1989, Douglas, 1990, Scannapieco et al., 1995, Vacca-Smith et al., 1996, Rogers et al., 1998, Rogers et al., 2001, Hannig et al., 2004). Estes autores sugeriram também que a enzima pode modular a colonização bacteriana no biofilme, pois atua na película adquirida como um receptor de alta afinidade para espécies de estreptococos que são colonizadores iniciais dos tecidos dentais, incluindo S. gordonii, S. mitis, S. parasanguis, S. crista, S. salivarius e S. sanguis. No biofilme, esta enzima facilita a hidrólise do amido e forneceria glicose adicional para o metabolismo de microorganismos em estreita proximidade com a superfície do dente (Scannapieco et al., 1993, Vacca-Smith et al., 1996, Rogers et al., 2001). Ainda, essa ligação da α-amilase com microorganismos orais em solução, contribui também para a depuração bacteriana (clearance) da cavidade oral (Scannapieco et al., 1993). Tem sido demonstrado que a presença de amido aumenta o potencial cariogênico da sacarose e o biofilme formado a partir dessa combinação exibiria diferenças em sua composição e estrutura, resultando na síntese de maior quantidade de glucosiltransferase B e polissacarídeos insolúveis. Isto aumentaria também a aderência de bactérias cariogênicas como S. mutans e levaria, consequentemente, a maior perda mineral durante os desafios cariogênicos (Ribeiro et al., 2005, Duarte et al., 2008).
A maioria dos estudos encontrados na literatura investiga a quantidade de proteínas totais e sua relação com a ocorrência de cárie (Kargul et al., 1994, Dodds et al., 1997, Tulunoglu et al., 2006, Roa et al., 2008, Preethi et al., 2010). Poucos estudos investigam especificamente a relação da enzima α-amilase com a ocorrência de cáries, sendo a literatura ainda inconclusiva (Fiehn et al., 1986, Liang et al., 1999, de Farias e Bezerra, 2003, Bardow et al., 2005, Vitorino et al., 2006, Shimotoyodome et al., 2007, Bhalla et al., 2010, Kejriwal et al., 2014, Singh et al., 2015). Os estudos que avaliam a saliva de crianças são escassos na
literatura (de Farias e Bezerra, 2003, Bhalla et al., 2010, Grychtol et al., 2015, Singh et al., 2015). Embora a enzima seja responsável pela quebra do amido, há também na literatura relato que pode haver um sinergismo entre a atividade de α-amilase e a presença de sacarose, de forma que a atividade da enzima no biofilme seria maior naquele formado na presença de sacarose (Dodds e Edgar, 1986). No entanto, não há na literatura estudos em crianças que tenham avaliado a atividade da enzima imediatamente após um desafio cariogênico considerando crianças com cárie e livres de cárie.
Além da participação dessa enzima como mediador no processo de cárie, também é descrito na literatura a possível participação da mesma na capacidade tampão realizada por proteínas, de modo que a α-amilase seria responsável por 35% da capacidade tampão de proteínas em faixa de pH de 4 a 5 (Cheaib e Lussi, 2013).
A partir do que foi exposto, torna-se relevante investigar como as enzimas α-amilase e AC VI se comportariam na saliva de crianças com cáries submetidos a um desafio cariogênico. Crianças com cárie estão sujeitas a modificações bioquímicas e microbiológicas importantes na saliva e no biofilme, decorrentes da alta exposição à sacarose, bem como a composição de proteínas da saliva e taxa de formação e aparência ultraestrutural da película difere entre dentes decíduos e permanentes (Nobre dos Santos et al., 2002, Parisotto et al., 2010, Grychtol et al., 2015). O estudo dos componentes salivares individualmente irá guiar o estudo da influência destes na comunidade microbiana de biofilmes e sua participação no processo de cárie (Nyvad, 2013). A modificação da atividade destas enzimas, ao ser o meio bucal exposto à sacarose também deve ser pesquisada em virtude de ser esse o principal substrato bacteriano, grande causador da cárie dental (Sheiham e James, 2015). Portanto os objetivos desta tese foram no Capítulo 1, investigar o comportamento da enzima AC VI, fluxo salivar, capacidade tampão antes e após um desafio cariogênico em crianças com cárie dental em um estudo longitudinal, e no Capítulo 2, Investigar o comportamento da enzima α-amilase, fluxo salivar e capacidade tampão antes e após um desafio cariogênico em crianças com cárie dental em um estudo transversal. Os capítulos serão apresentados em formato alternativo segundo a Resolução CCPG 001/2015 e encontram-se nas normas de publicação das revistas Archives of Oral Biology e Caries Research respectivamente.
2 ARTIGOS
2.1 Relationship between dental caries and salivary carbonic anhydrase VI activity, buffer capacity and flow rate – A longitudinal study in children
Artigo submetido ao periódico Archives of Oral Biology (Anexo 5)
Souza TRa, Zancopé BRa, Parisotto TMb , Rocha Marques M c, Nobre-dos-Santos Md*
a
DDS, MS, student of Department of Pediatric Dentistry, Piracicaba Dental School, University of Campinas, Piracicaba- SP, Brazil
b
DDS, MS, PhD of the Laboratory of Microbiology and Molecular Biology, Sao Francisco University Dental School, Bragança Paulista, SP, Brazil.
c
DDS, MS, Professor of Department of Morphology, Piracicaba Dental School, University of Campinas, Piracicaba- SP, Brazil.
d
DDS, MS, PhD, Professor of the Department of Pediatric Dentistry, Piracicaba Dental School, University of Campinas, Piracicaba- SP, Brazil.
Running Title: Salivary carbonic anhydrase VI and dental caries in children.
*
Corresponding Author: Prof. Marinês Nobre dos Santos, Av. Limeira, 901 Zip Code: 13414-903, Piracicaba-SP, Brazil, email: nobre@fop.unicamp.br, phone number: +55-19-21065290, Fax:+55-19-21065218
Abstract
Objective: To investigate the relationship among dental caries and salivary carbonic
anhydrase VI (CA VI) activity, buffering capacity (BC) and stimulated salivary flow rate (SSFR) in 48 to 78 month-old children. Design: After dental examination and caries diagnosis of 47 children, saliva was collected to evaluate SSFR, BC and CA VI activity before and after a 20% sucrose rinse at baseline and after one year of follow-up. Children were divided into three groups: caries free children (CF), children presenting caries lesions (CL), and children with arrested caries (AC). Presence of clinically visible biofilm in the upper incisors was verified. The activity of CA VI was quantified by zymography. The SSFR was expressed in mL/min and BC was measured using the Ericsson method. Wilcoxon and Kruskall-Wallis tests were used for comparisons. The Spearman correlation analysis was used for comparison between dental caries and independent variables and between BC and CA VI activity (α = 0.05). Results: At baseline, CA VI activity decreased significantly after the cariogenic challenge in CF children (p=0.037). No change in this parameter was noted for CL group at baseline and follow-up (p=0.825 and p=0.232, respectively). At follow-up, CA VI activity decreased significantly only at CF and AC group (p=0.028 and 0.027 respectively). The SSFR significantly increased after cariogenic challenge in all groups at baseline (p<0.05). At the follow-up SSFR was higher only at the AC group (p=0.001). BC decreased in all groups after cariogenic challenge at the baseline and follow-up (p<0.05) and values before rinse at baseline were negatively correlated to CA VI activity after rinse. At baseline, we found a moderate negative correlation between the salivary CA VI activity before and after sucrose rinse and dental caries in CL group (p=0.004 and 0.020 respectively). At follow-up, the same trend was noted only before sucrose rinse (p=0.013). Conclusion: In summary, this study demonstrated the participation of CA VI on the BC of saliva and suggest that children who had caries at baseline and continued to develop caries CA VI activity remains active after cariogenic challenge as a protection mechanism.
Introduction
Dental caries is a dynamic process caused by acids produced by bacteria inside a adherent biofilm that causes many cycles of demineralization and remineralization (Featherstone, 2008). The disease is one of the most common chronic disease of childhood, a serious public health problem in both developing and industrialized countries (Colak, Dulgergil, Dalli, & Hamidi, 2013). Among the several factors involved in the multifactorial etiology of dental caries, dietary sugars were recognized as the major cause of caries process because they provide a substrate for cariogenic oral bacteria to flourish and to generate enamel-demineralizing acids (Sheiham & James, 2015).
In the caries dynamic process, saliva is a protective factor against hard tissue loss and is essential for the maintenance of oral health. Saliva contains inorganic compounds and multiple proteins that affect conditions in the oral cavity and locally on the tooth surfaces. In addition, its neutralizing and remineralizing properties are important for healthy tooth structures (Dawes, 2003). In this regard, factors as the salivary flow rate and the buffering capacity act as protective in the carious process and have direct influence on the evaluation of the caries risk (Leone & Oppenheim, 2001; Tenovuo, 1997). To prevent the pH from decreasing to a critical level, saliva contains specific buffer mechanisms such as bicarbonate, phosphate and some protein systems, which have a buffering effect that neutralizes acids that oral cavity is exposed (Fejerskov & Kidd, 2007).
The main buffering system in stimulated saliva is the carbonic acid/bicarbonate buffer that is based on the equilibrium HCO-3 + H+ ↔H2CO3↔ CO2 + H2O, and is catalyzed by the isoenzyme carbonic anyhydrase VI (Breton, 2001). This enzyme is part of a group of isoenzymes that participate in a variety of physiological processes on the body that involve pH regulation, CO2 and HCO3- transport, ion transport, and water and electrolyte balance by catalyzing the reversible reaction described above (Kivela, Parkkila, Parkkila, Leinonen, & Rajaniemi, 1999a). CA VI is the only secreted isoenzyme of the CA family. It is secreted into saliva by serous acinar cells of the human parotid and submandibular glands (Parkkila et al., 1990). The presence of the enzyme was proved and quantified at the saliva and the presence of CA VI in the biofilm and its ability to connect to it and keep its activity in this place also was suggested. In this regard, early investigations suggested that in this site the enzyme catalyze the conversion of salivary bicarbonate and microbe-delivered hydrogen ions to
carbon dioxide and water (Leinonen, Kivela, Parkkila, Parkkila, & Rajaniemi, 1999; Parkkila, Parkkila, Vierjoki, Stahlberg, & Rajaniemi, 1993). Therefore, by this mechanism, this enzyme would protect teeth by catalyzing the most important buffer system in the oral cavity, thus accelerating the removal of acid (H+) from the local microenvironment of the tooth surface (Kivela, Parkkila, Parkkila, & Rajaniemi, 1999b). Later, it was suggested the role of CA VI in regulating dental biofilm pH (Kimoto, Kishino, Yura, & Ogawa, 2006).
The participation of CA VI on the caries process is not completely elucidated and literature shows conflicting results. Most of studies that investigated the role of CA VI on dental caries just determined the salivary concentration of CA VI, however, a high concentration of this isoenzyme in saliva does not necessarily mean that all enzyme is active in the middle (Aidar et al., 2013). Studies show a negative correlation between the CA VI salivary levels and caries and raised the hypothesis that CA VI present in saliva protected enamel surfaces from caries. (Kivela et al., 1999b; Szabo, 1974). However, a previous investigation found no evidence of the relationship between the concentration of the isoenzyme and dental caries (Ozturk et al., 2008). Later evidence demonstrated that the activity of CA VI was higher in saliva of preschool children with caries, highlighting the relevance of the isoenzyme being active in those subject who are frequently expose to cariogenic challenges (Frasseto et al., 2012). Although some of the previously cited studies have shown that CA VI isoenzyme is present in saliva, the results of its relationship with caries are conflicting and needs to be further investigated. Studies evaluating the behavior of this isoenzyme over time have not been reported in the literature. Thus, the aim of this follow-up study was to investigate the relationship among dental caries and salivary carbonic anhydrase VI activity, buffering capacity and stimulated salivary flow rate in 48 to 78 month-old children.
Materials and methods
Ethical Considerations
This study was approved by the Ethics Committee in Research of Piracicaba Dental School University of Campinas (UNICAMP) under protocol no. 014/2012. The Secretaria Municipal de Saúde of Piracicaba city of the State of Sao Paulo selected the two urban nurseries that could be used on the research. The procedures were explained to the parents of the subjects involved, and an informed written consent was obtained prior to the investigation.
At baseline and follow-up evaluations, children received a kit containing a toothbrush, fluoride toothpaste (1100 ppm F) and oral hygiene instructions. In addition, children who needed dental treatment were referred to receive comprehensive dental care at the Pediatric Dentistry Department of Piracicaba Dental School-University of Campinas.
Subjects
Three hundred children attending public pre-schools in the fluoridated (0.7 ppm F) urban area of Piracicaba, São Paulo state, were invited to take part in this study. At baseline, 104 children of both genders 53 (50.97 %) girls and 51 (49.03 %) boys of low socioeconomic level aging 48 to 78 months were allocated for the study. After a one-year of follow-up, 47 children (27 boys and 20 girls), mean age 72.3 months, remained in the cohort (55.8% of dropout rate) (Fig.1). This occurred because most of children, who were seven years old at follow-up, moved from their original pre-school and could not be found. After clinical examination, children were divided into three groups:
Caries free children, CF group (n=10): decayed, missing and filled surfaces plus early caries lesion=0 (dmft+ ECL), children who were caries-free at the beginning of the study and remained caries-free after one year;
Children presenting caries lesions, CL group (n=20): dmft+ECL ≥1. Children who had one or more caries lesions at the beginning of the study and continued to develop caries after one year;
Children with arrested caries or that had negative caries increment after one year, AC group (n=17).
Children with and without caries lesions were included in the study. The exclusion criteria of the study were children with systemic diseases, those who were under antibiotic therapy or taking medications for central nervous system diseases, children presenting communication or neuromotor difficulties as well as those with severe fluorosis, dental hypoplasia, children who refused the procedures or whose parents refused to sign the informed consent document were also excluded.
Calibration of the Examiner, Clinical examination and Caries Assessment
The examination considered all components of the World Health Organization diagnostic criteria and the early caries lesions (WHO+ECL) (Assaf, de Castro Meneghim, Zanin, Tengan, & Pereira, 2006). Dental examinations of each child were performed at baseline and after 1 year from the start of the study by only one examiner (T.R.S.) after calibration following cross-infection control measures. At first, clinical slides were used to train the examiner regarding the use of the WHO + ECL criteria. A clinical training session, using a gold standard for criteria, was held to achieve an acceptable level of agreement before the intraexaminer reliability assessment. The entire time spent on the calibration process (eg, theoretical discussions, training, and calibration exercises) was 30 hours. Intraexaminer reliability (Kappa calculation) regarding all components of the diagnostic criteria was assessed by re-examination of approximately 10% of children (both at baseline and at follow-up), with a 1-week-interval period. Kappa values at baseline and follow-up for the tooth surfaces were 0.82 and 0.80, respectively.
The examination was carried out with a focusable flashlight, a mirror and a ball-ended probe. Gauze was employed in order to dry or clean teeth, favoring the identification of early caries lesions. The units of evaluation used in the clinical examinations were d, m, f and s (decayed, missing and filled surfaces). Findings were recorded by a dental assistant.
Presence of visible biofilm examination
The presence of visible biofilm was observed on buccal surfaces of the four upper incisors by visual examination (Alaluusua & Malmivirta, 1994) and recorded in the clinical record as 0 for no visible biofilm and 1 for presence of visible biofilm.
Salivary Flow Rate and Buffering Capacity Determination
To avoid influence of the circadian rhythms, saliva samples were collected in the morning between 9 and 11 a.m., 2h after eating, drinking or chewing gum. Before sampling, children were left to relax for 5min. Each allocated children was instructed to chew a piece of parafilm weighing approximately 0.18g Parafilm® (Sigma Chemical Company, Missouri, USA) and to deposit whole saliva in a Falcon® tube (BD Biosciences, California, USA) for 5 min as previously described (Dawes & Kubieniec, 2004). If the secretion rate was low, the
collection was continued further for a maximum of 10 min and saliva was deposited in a sterile graduated ice-cooled container to prevent sample warming (Kirstila, Hakkinen, Jentsch, Vilja, & Tenovuo, 1998). All subjects were instructed to swallow at time zero. SSFR was calculated by measuring the total volume of saliva and dividing it by the collection time, and was expressed as mL/min (Ericsson & Hardwick, 1978). After the first saliva collection, a second collection of stimulated saliva was performed 5 minutes after a rinse with 5 ml of a 20% sucrose solution for 1 min (Frasseto et al, 2012). This procedure was performed to determine the effects that exposure of the oral environment to a cariogenic challenge would have on the salivary flow and buffering capacity, as well as on the activity of the CA VI isoenzyme.
After collection, saliva samples were immediately transported to the laboratory in a box containing ice sealed with plastic film to prevent the carbon dioxide elimination. BC of saliva was determined by Ericsson method (Ericsson, 1959). Thus, 0.5 mL of saliva was placed in a tube with 1.5 mL of HCl (0.005 mol/L), the tube was shaken mixed for 30 seconds using a vortex (AP 56, Phoenix) and a waiting period of 20 min was adopted for carbon dioxide elimination and the solution pH was measured. Buffering capacity was assessed using an electronic pH meter (Orion Analyzer Model 420A, USA).
After calculating the SSFR, and BC, saliva samples were centrifuged at 5.000 rpm for 10 min at 4oC, and stored in 2.0 mL microtubes, and were frozen at –40°C for later determination of CA VI activity.
Quantification of CA VI Activity in Saliva
The determination of CA VI activity was performed by the zymography method using a modified protocol (Aidar et al., 2013; Kotwica et al., 2006). After being thawed, 100 μL of saliva was added to 100 μL of Tris buffer. The solution was stirred before being placed on acrylamide gel at 30% and bisacrylamide at 0.8%. After that, 10 μL of this sample was placed in each channel of the gel, which remained for 1h: 50min at 140 V and at 4°C. After electrophoresis, the gel was stained with 0.1% bromothymol blue for 10 min. CA VI activity was observed after immersing the gel in distilled deionized water saturated with CO2. The gels were photographed, and images were quantified using the Image J software (Collins, 2007) was used to calculated the luminescence in area of the band, which expressed CA VI activity in numerical values (pixels/area).
Statistical analysis
The dependent variable was dental caries. The independent variables were: SSFR, BC and CA VI activity, before and after a sucrose rinse as well as presence of biofilm visible at the upper incisors. Data normality was checked using the Shapiro-Wilk test. Descriptive analysis by inferential statistics was performed and percentages, medians and interquatile ranges (IQR) were calculated for quantitative data of each independent variable before and after a 20% sucrose solution rinse (SSFR, BC and CAVI activity). Comparisons inside each group at baseline and follow-up before and after sucrose rinse were performed using the Wilcoxon test. Comparisons between the three groups were done using the Kruskal-Wallis test. Association between biofilm presence and dental caries at baseline and follow-up was determined using Fisher test. The Spearman correlation coefficient was calculated between SSFR, BC and CAVI activity and caries index at baseline and follow-up, also between BC and CA VI activity. We considered the 5% level of significance. Data were analyzed using the Statistical Package for Social Science 13.0 (SPSS Inc., IL, USA).
Results
The means and standard deviations of numbers of surfaces affected by caries at baseline and at follow-up in the studied population was 4.2 ± 4.8 and 5.3 ± 6.2 respectively (p=0.005, Wilcoxon test). The 1-year caries increment was 1.1 ± 2.5. In the CL group and AC group the mean numbers at baseline and follow-up were 6.5 ± 5.6 and 9.9 ± 6.5 / 3.94 ± 3.3 and 3.0 ± 3.2 respectively (p<0.005, Wilcoxon test). We also found a positive association between biofilm presence and caries at baseline and follow-up (p=0.037 and p=0.066 respectively for Fisher test).
Data of figure 2 demonstrate that at baseline after sucrose rinse, the SSFR significantly increased in the three investigated groups (p = 0.030; p=0.024 and p=0.002 for the CF group, CL group and AC group respectively). The same trend of result was found for the whole sample (p <0.001). At follow-up, results showed that the SSFR significantly increased only for the whole sample as well as for the AC group (p= 0.010 and p= 0.001, respectively).
Data of figure 3 demonstrate that at the baseline in all groups as well as in the whole sample, BC significantly decreased after sucrose rinse (p= 0.005, p= 0.001, p= 0.005 and p<0.001 for CF, CL, AC group and for the whole sample, respectively. At follow-up, we
noticed the same decreases in BC after sucrose rinse (p= 0.047, p= 0.003, p=0.001, p<0.001 respectively for the same).
Table 1 shows that at baseline, a significant decrease in CA VI activity occurred after sucrose rinse for the whole sample as well as for the CF group (p= 0.015 and p= 0.037 respectively). On the other side, at this time no change in this parameter was noted for the CL group (p= 0.825). At follow-up, after sucrose rinse, the CA VI activity was significantly lower for the whole sample, as well as for the CF and AC groups (p=0.03, p=0.028 and p=0.027 respectively). However, no change in CA VI activity was found for the CL group (p=0.232) (Table 2). We could not find any difference among groups related to CA VI activity and its variation at baseline and follow-up (Table 1 and 2).
Correlations between dental caries and variable characteristics at baseline and follow-up are shown in Table 3. The results demonstrate that at baseline, variables that showed a significant negative correlations with dental caries were BC after rinse (r = -0.345 and p = 0.017) and CA VI activity before rinse (r = -0.305 and p = 0.037). At follow-up, BC after sucrose rinse was the only variable that showed a negative correlation with caries. (r = -0.308 and p = 0.038).
Table 4 shows that for the CL group, there is a significant negative moderate correlation between dental caries and CA VI activity before and after sucrose rinse at baseline (r = -0.609 p = 0.004 and r=-0.516 p=0.020 respectively). At follow-up a significant negative correlation between dental caries and CA VI activity before sucrose rinse was also found (r = -0.545 p = 0.013).
Table 5 reveals the correlations between BC and CA VI activity before and after sucrose rinse at baseline and follow-up. A negative correlation between BC before sucrose rinse and CA VI activity before sucrose rinse was detected at follow-up (r=-0.366, p=0.011) but not at baseline. This table also reveals a negative correlation between BC after sucrose rinse and CA VI activity before sucrose rinse only at follow-up (r=-0.378, p=0.009).
Discussion
Our study investigated for the first the time behavior of CA VI activity in whole saliva of children before and 5 minutes after a cariogenic challenge in caries-free children, as well as in those with caries and with arrested caries after one year of follow-up. The results of the present study showed that the CA VI activity exhibits a different behavior when submitted
to a cariogenic challenge whether children were caries-free, had only arrested caries or had caries.
At baseline and follow-up, the CF group as well as the AC group at the follow-up showed significant decreases in the CA VI activity after sucrose rinse. These results were expected and can partially be explained if we consider that caries-free children as well as those having arrested caries are less frequently submitted to pH drops as a consequence of low acid production after the cariogenic challenge which decreased enzyme activity, since the acid is also a substrate for the reaction. Carbonic anhydrase VI catalyzes the reaction of HCO-3 + H+ ↔H2CO3↔ CO2 + H2O in both directions and it is possible that it may neutralize the media (Leinonen et al., 1999). This result is in accordance with the negative correlation between the BC before rinse and activity CA VI after rinse and at the baseline and the supply of H+ ions as a substrate for the reaction catalyzed by CA VI. On the other hand, early investigations found no association between salivary pH, BC and CA VI concentration in saliva (Kivela et al., 1997; Parkkila et al., 1993). However, it is important to notice that it is known the CA VI isoenzyme catalyzes the reaction that balances pH in the oral cavity after a cariogenic challenge, so the results of this study was expected.
Another result of this study was that at the two periods of study, the CL group (dmft > 0) showed no change in CA VI activity after sucrose rinse. Children having caries are frequently exposed to high daily sugar consumption and it is known that this sugar consumption pattern is significantly correlated with early childhood caries (Nobre dos Santos, Melo dos Santos, Francisco, & Cury, 2002; Parisotto et al., 2010). In the presence of a sugar-rich diet and a greater acid formation by metabolism of dental biofilm microbiota in this group, there is a possibility that salivary CA VI activity remained unchanged after the sucrose rinse to provide a higher protection against dental caries. The suggested mechanism would be that that in these individuals salivary CA VI would neutralize greater amounts of acid mainly in the form of latic, acetic, formic and propionic produced by the microbial metabolism in the mouth and over dental surfaces. This acid neutralization would be accomplished via conversion of salivary bicarbonate and microbe-delivered hydrogen ions to carbon dioxide and water catalyzed by salivary CA VI (Leinonen et al., 1999). In line with this assumption, in this group, we found a moderate negative correlation between CA VI activity before as well as after sucrose rinse and dental caries (Table 4). A further explanation for this finding, could be that if the CO2 + H2O ↔ H+ + HCO3– reaction is fueled by HCO3- provided by
salivary CA II supply and H+ delivery by the microbial metabolism of carbohydrates, the reaction would work in a reverse way by neutralizing the salivary pH and this fact may have a role in children with caries. And this was confirmed by the essential feature of this buffer system under the conditions prevailing in the oral cavity is the phase conversion of carbon dioxide from a dissolved state into a volatile gas (Kivela et al., 1999a). In the other side, in caries-free subjects and in those who had arrested caries at baseline and at follow-up, after the cariogenic challenge, there was a significant reduction in the CA VI activity probably as consequence of a low acid production of in the oral environment since these individuals are less frequently exposed to cariogenic carbohydrates and consequently, to regular pH falls in saliva and dental biofilm (Nobre dos Santos et al., 2002, Parisotto et al., 2010). In this way, acid buffering in the oral environment provided by CA VI activity would not be so necessary in these individuals. In this regard, previous investigations suggested that the isoenzyme participates not only in preventing caries development by always maintaining the pH of oral cavity at a level higher than the critical one, but also appears to be active during the occurrence of a cariogenic challenge in individuals with the disease already installed. (Frasseto et al., 2012; Leinonen et al., 1999).
Our study, did not notice any difference in the results of CA VI activity among groups neither before nor after the cariogenic challenge. Regarding before rinse data our results are in accordance with Ozturk et al. (2008) who also did not find any difference in CA VI concentration between caries and caries-free young adults. However, different findings were obtained by Frasseto et al. (2012). These authors found a higher CA VI activity in the caries group than in the caries-free group before sucrose rinse (p=0.0516). Concerning CA VI activity after sucrose rinse, our results are in line with Frasseto et al. (2012).
Another result of this study was that at baseline and at follow-up, there was no difference among groups concerning the variation of CAVI activity. Our data are not in line with the results found by Frasseto et al. (2012), who detected that variation of CA VI activity was significantly higher in the CL group than in the CF group. A possible explanation for these findings could be the large inter-individuals variation of CA VI concentration and activity as pointed out in several studies (Frasseto et al., 2012; Kivela, Laine, Parkkila, & Rajaniemi, 2003; Parkkila, Parkkila, & Rajaniemi, 1995).
Based on the findings regarding the CA VI behavior in the oral environment, the isoenzyme should not be interpreted as a factor that favors the decay process, but as protective
salivary protein acting in an attempt neutralize the pH of acid produced as previously demonstrated by Kimoto et al. (2006). This mechanism would be most important especially in subjects having caries to whom the enzyme would be more active after a cariogenic challenge, as a catalyst agent in the buffering reaction of bicarbonate in saliva. The findings of this study suggest that the CA VI behavior did not change with pH drop in the oral cavity at CL group. In line with this thought, the recent data of a genetic study that suggest that salivary CA VI plays an important role in protecting teeth from caries (Li, Hu, Zhou, Xie, & Zhang, 2015).
The results of the present study also showed a moderate negative correlation between CA VI activity before rinse and dental caries at the baseline as well as at follow-up in the CL group (Table 5). There is a possibility that in these subjects the higher enzyme activity would act better to control oral pH under normal conditions before and after the cariogenic challenge, in the oral cavity. These results are in agreement with Kivela et al. (1999b), who claimed that this correlation with CA VI concentration was most significant in subjects with poor oral hygiene. In line with this assumption, our results showed a significant association between dental caries and biofilm presence. In the other side, our results differed from those obtained by Ozturk et al. (2008) and Frasetto et al., (2012) who did not find any correlation between dental caries and CA VI concentration and activity respectively.
Saliva is believed to be one of the most important host factors and an essential mediator controlling the speed and direction of the cariogenic pathway (Gao, Jiang, Koh, & Hsu, 2016). Our study also showed that at baseline the SSFR increased significantly after sucrose rinse in the three groups. The results are in line Frasseto et al. (2012). However, at follow-up this change was noted only in the AC group. These findings can be explained if we consider the mechanical and gustatory stimulation promoted by rinse and the stimulus of the salivary glands provided by sucrose (Proctor, 2016). These results also are in accordance with found by Dawes & Kubieniec (2004). We did not found any difference among groups regarding SSFR at baseline and follow-up and any correlation between caries and SSFR at baseline or at follow-up. In line with this assumption, previous studies have shown that in individuals with normal salivary flow rates, the relationship between salivary flow and caries has little or no predictive value for the occurrence of disease (Lenander-Lumikari & Loimaranta, 2000).
Salivary pH and buffering capacity are known to be central factors protecting teeth from caries and could be considered a moderate risk factor for its prevalence and incidence
(Gao et al., 2016; Kivela et al., 1999b). Concerning BC, we noticed a significant decrease in all groups at baseline and follow-up after sucrose rinse. This result is in line with those obtained by Frasseto et al. (2012) for biofilm pH after sucrose rinse in caries and caries-free children. Moreover, we also found a significant negative correlation between BC after sucrose rinse and dental caries at baseline as well as at follow-up. For baseline data, similar results were found by Kivella et al. (1999b) and are in line with previous studies (Kuriakose, Sundaresan, Mathai, Khosla, & Gaffoor, 2013; Ruiz Miravet, Montiel Company, & Almerich Silla, 2007; Singh et al., 2015; Yildiz, Ermis, Calapoglu, Celik, & Turel, 2016). However, these authors did not perform sucrose rinse in their investigation. We did not find any difference among groups concerning BC. For baseline data, these results are in agreement with previous investigations (Peres et al., 2010; Yarat et al., 2011).
In summary, this study suggests that the enzyme CA VI provides a protective role when the oral cavity environment is submitted to cariogenic challenge. In addition, a low CA VI activity showed to correlate with caries prevalence before cariogenic challenge mainly in caries children. Our findings demonstrate the importance of this enzyme as a participant of the mouth physiology in controlling saliva after cariogenic challenges. In conclusion, this study demonstrated that CA VI isoenzyme remains active in saliva of children with caries after cariogenic challenge with sucrose and suggests the participation of CA VI on the BC of saliva.
Funding
The study was supported by FAPESP (2012/02516-1 and 2012/15834-1).
Competing Interests
The authors reported no conflict of interest. The authors alone were responsible for the content and the writing of the paper.
Ethical Approval
The protocol was approved by the local Bioethics Committee of Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil (Protocols #014/2012).
Acknowledgements
This paper was based on a thesis submitted by the first author to Piracicaba Dental School, University of Campinas, in partial fulfillment of the requirements for a DDS degree in Dentistry (Pediatric Dentistry area). This study was supported by FAPESP (2012/02516-1 and 2012/15834-1). We thank the Secretary of Education and Health of Piracicaba-SP/Brazil for collaborating with this research. We specially thank the volunteers and their parents for participating in this research.
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