Tese mestrado Karina Vieira de Barros Munhoz [PDF]

KARINA VIEIRA DE BARROS MUNHOZ

EFEITO DE DIETAS RICAS EM ÓLEO DE PEIXE OU ÓLEO DE SOJA SOBRE MEDIADORES INFLAMATÓRIOS E DANO OXIDATIVO DO DNA NA COLITE EXPERIMENTAL

Tese apresentada à Universidade Federal de São Paulo, como requisito parcial para obtenção do Título de Mestre em Ciências.

São Paulo 2009 1

KARINA VIEIRA DE BARROS MUNHOZ

EFEITO DE DIETAS RICAS EM ÓLEO DE PEIXE OU ÓLEO DE SOJA SOBRE MEDIADORES INFLAMATÓRIOS E DANO OXIDATIVO DO DNA NA COLITE EXPERIMENTAL

Tese apresentada à Universidade Federal de São Paulo, como requisito parcial para obtenção do Título de Mestre em Ciências.

Orientadora: Profa. Dra. Vera Lucia Flor Silveira

São Paulo 2009

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Barros, KV Efeito de dietas ricas em óleo de peixe ou soja sobre mediadores inflamatórios e dano oxidativo do DNA na colite experimental. – Karina Vieira de Barros Munhoz – São Paulo, 2009.

Tese

(Mestrado)

–

Universidade

Federal

de

São

Paulo.

Departamento de Fisiologia da Nutrição. Programa de Pós-graduação em Nutrição.

1.Doença inflamatória intestinal. 2.Ácidos Graxos Poliinsaturados w6 e w-3. 3. Dano oxidativo de DNA. 4.Citocinas.

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Este trabalho foi realizado na Disciplina de Fisiologia da Nutrição, Departamento de Fisiologia da Universidade Federal de São Paulo, sob a orientação da Profa. Dra. Vera Lúcia Flor Silveira

Apoio Financeiro da Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP) e da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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“Ser mestre é um ato de fé. Fé na possibilidade de mudar o mundo educando, fé no indivíduo, fé na supremacia da riqueza intelectual. Ser mestre é um ato de amor. Porque a entrega de si está implícita na tarefa, porque se dá com as mãos cheias sem esperar retribuição. Ser mestre é ser sonhador. Crer, mais além desta época frívola e céptica, no espírito do homem. E crer que algum dia, ao final do caminho, poderemos transferir esta tocha a um discípulo, outro sonhador”.

Lídia Maria Riba

Dedico este trabalho:

Aos meus pais, minha irmã e meu marido pelo esforço, dedicação e compreensão, em todos os momentos desta e de outras caminhadas.

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AGRADECIMENTOS

À Profa. Dra. Vera Lucia Flor Silveira, minha orientadora, pelo apoio, paciência, credibilidade e compreensão que me proporcionou crescimento pessoal e profissional. Por suas lições e seus exemplos, meu sincero agradecimento!

Ao Prof. Dr. Carlos Augusto Real Martinez pela amizade, carinho e dedicação confiando na minha capacidade e incentivando meu crescimento.

Aos meus amigos e companheiros de trabalho: Gilclay Gomes de Abreu e Roberta Araújo Navarro Xavier. Nada na vida conquistamos sozinhos. Sempre precisamos de outras pessoas para alcançar os nossos objetivos. Muito obrigada pelo apoio e pela paciência durante a realização dos experimentos, pelos momentos de alegrias, pelas risadas e pelas valiosas conversas.

À Profa. Dra. Luciana Pellegrini Pizani e ao Prof. Dr. Andrea Bottoni pela confiança, incentivo inicial e amizade.

À Profa. Dra. Claudia Oller do Nascimento por disponibilizar auxílio técnico e científico nos momentos de necessidade.

À minha querida amiga e professora de inglês Ivana Ribeiro Cunha, a quem sou eternamente grata pelos conhecimentos da língua inglesa.

Aos Professores Dr. Marcelo Lima Ribeiro, Dra. Patrícia Oliveira Carvalho e Dra.

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Alessandra Gambero pela colaboração no desenvolvimento deste trabalho.

Ao amigo André Luis Lacerda Bachi pelos freqüentes auxílios no entendimento da imunologia. Muito obrigada!

Ao amigo Alexandre Basile, parceiro de todas as horas, que sempre esteve disposto a me ajudar nos momentos de necessidade. Obrigada Alemão!

Aos amigos do laboratório pela alegre convivência e sugestões, dividindo conhecimentos e experiências. Ana Barbosa Marcondes de Mattos, Caio Sussumu de Macedo Motoyama, Carla Rodrigues de Carvalho, Carolina Biz Rodrigues Silva, Cristiane de Oliveira, Elisabete dos Reis Carneiro, Fábio Santos de Lira, Iracema Senna de Andrade, Juliane Costa Silva Zemdegs, João Felipe Mota, José Cesar Rosa Neto, Regina Lúcia Harumi Watanabe, Ricardo Eguchi, Roseli Sandra Silva e Vinícius José Baccin Martins.

Às professoras Dra. Eliane Beraldi Ribeiro, Dra. Kelse Tibau de Albuquerque, Dra. Lila Oyama e Dra Mônica Marques Telles pelo agradável convívio no Laboratório de Fisiologia da Nutrição da UNIFESP.

A todos os funcionários do laboratório de Fisiologia da Nutrição, em especial à Ana Lúcia Castro Santos e Mauro Cardoso Pereira pela dedicação e responsabilidade essenciais para a conclusão deste trabalho.

Aos meus familiares que sempre me apoiaram. A minha mãe Wilma Maria Vieira de

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Barros e ao meu pai Pedro Paulo de Barros, meus principais motivos de orgulho, que não me deram somente a vida, mas principalmente a minha educação e condições de estudo além de incentivo incondicional na minha formação pessoal e profissional. A minha irmã Ana Clara Vieira de Barros por sempre torcer por mim e estar sempre presente.

Ao meu marido Eduardo Della Valle Munhoz, por se orgulhar e acreditar em mim sempre. Muito obrigada pelo incentivo!

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Para sempre ...

“Há homens que lutam um dia e são bons. Há outros que lutam um ano e são melhores. Há os que lutam muitos anos e são muito bons. Porém, há os que lutam toda a vida. Esses são os imprescindíveis.” Bertolt Brescht

Aprendi ... Não tenho um caminho novo. O que tenho de novo é um jeito de caminhar.” Thiago de Melo.

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RESUMO Temos demonstrado, em nosso laboratório, que dietas lipídicas enriquecidas com óleo de peixe ou óleo de soja apresentam efeito antiinflamatório associado a alterações em mediadores inflamatórios, como corticosteróides, bradicinina, calicreína, óxido nítrico e citocinas, em modelo de inflamação aguda. Embora o consumo de dietas enriquecidas com óleo de peixe tem também sido associado à diminuição da inflamação crônica, dietas hiperlipídicas parecem aumentar o risco de desenvolvimento da colite ulcerativa e de câncer de colon. Neste trabalho, verificamos se dietas hiperlipídicas ou normolipídicas, enriquecidas com óleo de peixe, óleo de soja ou com a mistura desses podem alterar o processo inflamatório, a liberação de mediadores inflamatórios e o dano oxidativo do DNA na colite experimental, induzida por sulfato de sódio dextran (DSS). Para isto, ratos machos Wistar, recém-desmamados foram divididos em 6 grupos, alimentados durante 47 dias com as dietas experimentais; 3 grupos foram alimentados com dietas hiperlipídicas (20%), ricas em óleo de soja (HS), óleo de peixe (HP) ou mistura de óleo de soja e peixe (HSP) e 3 grupos foram alimentados com dietas normolipídicas (5%) enriquecidas com óleo de soja (S), óleo de peixe (P) ou mistura de óleo de soja e peixe (SP). A dieta S foi o controle tanto dos grupos hiperlipídicos como dos grupos normolipídicos. A colite foi induzida, em todos os animais, no dia 36, com 3% de DSS na água de beber por 7 dias (dia 36 ao 42). No 43º dia o DSS foi descontinuado e no 48º dia os animais foram sacrificados, após jejum alimentar de 24 horas. Durante a indução da colite foi avaliado diariamente o índice de atividade da doença (IAD). Foram coletadas amostras sanguíneas para dosagem de corticosterona, o fígado para análise da incorporação dos ácidos graxos

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e o cólon distal que foi dividido em fragmentos para análise histológica, dano oxidativo de DNA, dosagem da atividade da enzima mieloperoxidase (MPO) e das concentrações teciduais das citocinas IL-4, IFNγ e IL-10. Nos grupos alimentados com dietas hiperlipídicas não foram encontradas diferenças nas concentrações de IL-4 e INF-γ, MPO, corticosterona, índice de inflamação, comprimento do cólon e IAD. No entanto, a dieta HP aumentou as concentrações de IL-10 em relação à dieta HS e somente a dieta HSP aumentou a razão IL-10/IL-4 (citocina antiinflamatória / proinflamatória) associada à diminuição do dano de DNA. Esses dados demonstram que dietas hiperlipídicas, ricas em óleo de soja ou peixe, não exacerbam a colite experimental em relação ao controle normolipídico (S), e que a mistura desses óleos tem efeito benéfico no equilíbrio das citocinas e na manutenção da integridade do DNA. Nos grupos alimentados com dietas normolipídicas não foram observadas diferenças nos níveis de corticosterona, IFN-γ e IL-4. Somente a dieta SP diminuiu o IAD, a MPO e o índice de inflamação, além de aumentar a IL-10 e proteger contra o dano de DNA em relação ao controle (S). A dieta P promoveu um efeito intermediário, não sendo diferente do controle e da dieta SP, em relação à gravidade da doença, porém aumentou os níveis de IL-10 e diminuiu o dano de DNA em relação à dieta controle (S). Adicionalmente, foi observado uma alta correlação entre o aumento de IL-10 e a diminuição do dano de DNA. Esses dados demonstram que a dieta normolipídica SP reduz a injúria colônica e o dano de DNA e sugerem que essa dieta (SP) pode contribuir como terapia complementar na colite ulcerativa para diminuir o uso de drogas antiinflamatórias e prevenir o câncer de colon.

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ÍNDICE

1.0 Introdução ....................................................................................................... 13 1.1 Justificativa ........................................................................................................ 24

2.0 Objetivos .......................................................................................................... 26

3.0 Material e Métodos .......................................................................................... 27 3.1 Animais e tratamento ........................................................................................ 27 3.2 Indução da colite experimental ......................................................................... 28 3.3 Preparações das dietas e armazenamento ...................................................... 29 3.4 Consumo alimentar e peso corporal ................................................................. 31 3.5 Coleta das amostras ......................................................................................... 31 3.6 Análise histológica ............................................................................................. 32 3.7 Escore do índice de gravidade da doença ....................................................... 33 3.8 Atividade da enzima mieloperoxidase (MPO) ................................................... 34 3.9 Concentrações de citocinas no tecido colônico ................................................ 35 3.10 Ensaio cometa ................................................................................................ 36 3.11 Incorporação de ácidos graxos no fígado ....................................................... 37 3.12 Determinação de corticosterona plasmática ................................................... 38 3.13 Análise estatística ........................................................................................... 39

4.0 Resultados e Discussão................................................................................. 40 Artigo I ..................................................................................................................... 41 Artigo II .................................................................................................................... 70

5.0 Referências Bibliográficas ................................................................................. 94

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1 - INTRODUÇÃO As doenças inflamatórias intestinais (DII) são afecções crônicas do trato gastrointestinal (TGI), que geralmente se referem a duas condições, retocolite ulcerativa ou colite ulcerativa e doença de Crohn (Galvez et al, 2006). As DIIs caracterizam-se por diarréia crônica, má absorção, disfunção da barreira mucosa e processo inflamatório intestinal, sendo clinicamente incuráveis (Benedetti & Plum, 1996). A colite ulcerativa engloba um espectro de inflamação difusa, contínua e superficial do cólon, que começa no reto e pode se estender até o nível proximal. Já a doença de Crohn caracteriza-se por inflamação transmural assimétrica que afeta qualquer porção do TGI, desde a boca até o ânus (Benedetti & Plum, 1996). Embora muitos progressos tenham sido feitos no entendimento das DIIs, sua etiologia ainda não está totalmente elucidada, mas acredita-se que haja o envolvimento de fatores imunes, genéticos e ambientais. (Laroux et al, 2001; Cheon et al, 2006; Sainathan et al, 2008). Alguns trabalhos têm sugerido que as DIIs representam uma resposta inapropriada e exagerada do sistema imune da mucosa intestinal à microflora intestinal normal, em indivíduos geneticamente suscetíveis, que pode ser atribuída, em parte, ao desequilíbrio entre as células T efetoras (T ef) e células T reguladoras (T reg). (Ma et al, 2007; Sanchez-Muñoz et al, 2008). As células T efetoras são os linfócitos T auxiliares (Linf T CD4+) e os linfócitos T citolíticos (Linf T CD8+) ativados durante a resposta imunológica adquirida ou adaptativa. As células T auxiliares secretam citocinas, cuja função é estimular a proliferação e a diferenciação das células T, assim como de outras células incluindo linfócitos B, macrófagos e outros leucócitos (Sainathan et al, 2008; Sanchez-Muñoz et al, 2008). Os linfócitos T citolíticos destroem células que produzem antígenos

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estranhos, como as células infectadas por vírus ou outros microorganismos intracelulares. Já as células T reguladoras são células capazes de bloquear a ativação e a função dos linfócitos T efetores (Abbas & Lichtman, 2005). Alguns estudos indicam que a ação supressora dessas células está ligada à secreção de citocinas imunossupressoras, como Interleucina-10 (IL-10) e o Fator de crescimento transformador beta (TGF-β). O TGF- β inibe a proliferação de células T e B, enquanto que a IL-10 inibe a ativação de macrófagos e é antagonista do principal fator de ativação de macrófagos, o Interferon gama (IFN-γ) (Sanchez-Muñoz et al, 2008). Nas DIIs, a resposta imune inata também tem um importante papel. Essa resposta é a linha de defesa inicial do sistema imunológico, onde participam células fagocitárias, células natural killers, proteínas do sangue, incluindo frações do sistema complemento e outros mediadores da inflamação como as citocinas (Abbas & Lichtman, 2005). Citocinas são polipeptídeos produzidos principalmente pelas células imune que facilitam a comunicação entre células, estimulam a proliferação de células efetoras específicas para antígenos e medeiam a inflamação sistêmica e local nas vias endócrinas, parácrinas e autócrinas (Sanchez-Muñoz et al, 2008). As células dendríticas e os macrófagos ativados secretam várias citocinas, que regulam a resposta inflamatória. Uma vez secretadas, essas citocinas promovem a diferenciação de células T, ativando a resposta imune adaptativa (Abbas & Lichtman, 2005). Os linfócitos T auxiliares ou células T CD4+ podem se diferenciar em subpopulações de células T efetoras que produzem conjuntos diferentes de citocinas

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e, portanto, desempenham funções efetoras distintas. As subpopulações mais bem definidas de células T efetoras são as células T helper do tipo 1 (Th1) e do tipo 2 (Th2) (Fuss et al, 2004; Abbas & Lichtman, 2005). O IFN-γ está associado ao padrão Th1, enquanto IL-4 e IL-5 associam-se as células Th2. Hoje já está claro que células individuais podem expressar várias misturas de citocinas, e que pode haver muitas subpopulações com padrões heterogêneos de produção de citocinas. Entretanto, as reações imunes crônicas são freqüentemente dominadas por uma das duas populações Th1 ou Th2 (Kampen et al, 2005). Essas subpopulações mostram diferenças na expressão de vários receptores de citocina, e essas diferenças podem refletir o estado de ativação da célula, além de determinar as suas funções efetoras e participar no desenvolvimento e expansão das respectivas subpopulações (Abbas & Lichtman, 2005). Nas DIIs ocorre um desequilíbrio entre a resposta T reguladoras / Th1, Th2. A falta da regulação apropriada das células T ou a super produção de células T efetoras está relacionada ao desenvolvimento e exacerbação das DIIs (Zhang et al, 2005; Sanchez-Muñoz et al, 2008). A IL-10 é considerada uma citocina imunorreguladora que tem impacto tanto na resposta imune inata quanto na adquirida. Animais knockout para IL-10 desenvolvem colite espontaneamente, sendo que 30 a 60% desses animais apresentam carcinoma invasivo de cólon entre os 3 e 6 meses de idade (McCafferty et al, 2000; Hegazi et al, 2006). Esses animais apresentam duas características importantes: (1) aumento da permeabilidade intestinal em uma fase precoce da vida e antes do início da doença, e (2) desenvolvimento de colite, dependente dos fatores microbiológicos presentes no lúmen intestinal. Tais características sugerem que a

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colite observada nesses animais pode se desenvolver como conseqüência da elevada permeabilidade intestinal que propicia aumento dos agentes luminais no sistema imune mucosal (Arrieta et al, 2008). Alguns estudos têm demonstrado o papel da IL-10 na manutenção da homeostase mucosal gastrointestinal. O mecanismo pelo qual essa citocina regula a inflamação mucosal é provavelmente multifatorial, porém, está associado à diminuição da apresentação de antígeno (McCafferty et al, 2000; Hegazi et al, 2006), aumento da liberação de Interferon gama (IFN-γ) e de IL-12, citocina que inibe a diferenciação dos linfócitos T em linfócitos Th1 (Rennick & Fort, 2000). Há fortes evidências de que a IL-10 promova a diferenciação e aumento da atividade das células T regulatórias (Hegazi et al, 2006). Estudos in vitro têm demonstrado que a administração de IL-10 diminui a liberação de citocinas pró-inflamatórias de células mononucleares na lâmina própria de pacientes com doença de Crohn. Em adição, altas doses de IL-10, administradas por via intraperitoneal em camundongos com colite induzida por ácido Trinitro benzeno sulfônico (TNBS Trinitrobenzenesulphonic acid), são capazes de restaurar a tolerância de células mononucleares da lâmina própria (Duchmann et al, 1996). De acordo com Galvez et al (2006) as DIIs são conseqüentes a um aumento da síntese e liberação de diferentes mediadores pró-inflamatórios, incluindo eicosanóides, fator ativador de plaquetas (PAF), citocinas, espécies reativas de oxigênio (ROS), metabólitos nitrogenados, ativação do fator nuclear de transcrição kB (NF-KB) e inibição da apoptose. Esses mediadores contribuem para a cascata patogênica que inicia e perpetua a resposta inflamatória do intestino (Camuesco et al, 2004; Sanchez-Muñoz et al, 2008,).

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Embora o mecanismo envolvido nessa resposta não esteja completamente elucidado, a ativação de macrófagos teciduais bem como o recrutamento e ativação de

leucócitos

fagocíticos

adicionais

(neutrófilos,

eosinófilos

e

monócitos),

principalmente neutrófilos (Nieto et al, 1998; Camuesco et al, 2006), tem papel primordial na resposta inflamatória do intestino (Pavlick et al, 2002; Galvez et al , 2006). Este infiltrado inflamatório aumentado é acompanhado por extensiva injúria mucosal e transmural, incluindo aumento da permeabilidade vascular, destruição da matriz extracelular e dano celular epitelial (Nieto et al, 1998). Um dos principais mecanismos de destruição tecidual é o estresse oxidativo, que ocorre devido à excessiva síntese e liberação de ROS pelos leucócitos (McCord et al. 2000, Nieto et al. 2002). Embora a formação desses radicais seja essencial para a defesa do hospedeiro contra a infecção bacteriana, sua continua superprodução, durante o processo inflamatório, pode causar extensa destruição tecidual (Roessner et al, 2008). Pacientes com DIIs parecem ter alteração na função da barreira intestinal. A atividade da doença é associada ao influxo de neutrófilos dentro do epitélio mucosal e, subseqüentemente dentro do lúmen intestinal, resultando nos abscessos de criptas (Arita et al, 2005; Bernstein et al, 2006; McGuckin et al, 2009). Doentes portadores de DIIs, e em particular de colite ulcerativa, tem risco de desenvolvimento de neoplasias 10 vezes maior do que a população geral, indicando que a inflamação intestinal crônica é um fator de risco importante para o desenvolvimento de câncer de cólon (Gommeaux et al. 2007). Alguns estudos têm demonstrado que o risco de desenvolvimento de câncer aumenta exponencialmente com a duração da doença e com a extensão e intensidade do processo inflamatório

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na mucosa intestinal (Burstein & Fearon, 2008). O processo de carcinogênese parece envolver uma seqüência de eventos, onde o epitélio cronicamente inflamado e hiperplásico progride inicialmente para focos de displasia plana, adenoma e finalmente para o adenocarcinoma. A inflamação não controlada é associada ao estresse oxidativo e ao dano celular oxidativo. Durante a proliferação das células, lesões oxidativas de DNA induzem mutações que são comumente observadas na oncogênese e nos genes supressores de tumor como o p53 (Seril et al, 2003; Gommeaux et al, 2007). É provável que as células da mucosa cólica, persistentemente

submetidas

a

agentes

oxidantes,

sofram

dano oxidativo

progressivo em seu DNA, o que pode ocasionar mutações em genes supressores de tumor (p53), oncogênese (k-ras) e em genes que codificam as proteínas de reparo (MSH2 e MLH1) (Gommeaux et al, 2007). A iniciação da carcinogênese é causada por uma alteração irreversível no DNA, como a reação dessa molécula com substâncias carcinogênicas. Assim, mecanismos de detoxicação de carcinógeno, reparo do DNA e eliminação das células que tenham DNA modificado (por apoptose, por exemplo) são importantes para a proteção contra a iniciação do câncer (Brown et al, 1994). Para que a iniciação ocorra, é necessário que haja não só a modificação do DNA, mas também a sua replicação e proliferação celular, de modo que a mutação inicial possa se fixar. A maioria dos cânceres humanos é originária de células epiteliais (carcinoma), pois as mesmas estão expostas aos carcinógenos (presentes no ar ou alimentos) e se proliferam rapidamente (Bartsch et al, 1996). Em geral, os carcinógenos são substâncias eletrofílicas ou são metabolizados para as mesmas durante o seu processo de detoxicação. Tais substâncias são atraídas por moléculas com alta densidade eletrônica, como por exemplo, as bases 18

do DNA, as quais acabam se ligando e levando a formação de adutos (Bartsch et al, 2006). A base do DNA mais suscetível a esse tipo de ataque é a guanina, mas já foram relatados adutos formados em outras bases. Sendo formados no DNA por mecanismos químicos específicos, tais adutos podem levar a mutações em protooncogênese ou em genes supressores de tumor e iniciar o processo de carcinogênese (Lehman et al, 1994; Kinzler et al, 1996). É bem estabelecido que a inflamação facilita a progressão de células normais para células malignas, pela produção de citocinas pró-inflamatórias como o Fator de necrose tumoral (TNF), IL-1, IL-6, IL-23 e espécies reativas de oxigênio e nitrogênio (Bartsch et al, 2006; Roessner et al, 2008). A grande quantidade de citocinas e fatores de crescimento liberados durante a inflamação, pelas células do sistema imune e células não imunes, podem influenciar o processo de carcinogênese (Fantini et al, 2008). Esses mediadores ativam o NF-KB, a óxido nítrico sintase indutível (iNOS) e a ciclooxigenase do tipo 2 (COX-2), que estão associados ao retardo ou supressão da apoptose das células epiteliais intestinais e modulação da angiogênese (Chapkin et al, 2007). A apoptose, morte celular programada, é o mecanismo pelo qual o intestino elimina células com dano de DNA irreparável, e a inibição dessa resposta é uma das características do câncer de cólon (Bancroft et al, 2003). Alguns estudos têm demonstrado associação entre suplementação dietética de óleo de peixe e aumento da apoptose no topo das criptas colônicas, região onde geralmente os tumores e pólipos se desenvolvem (Paulsen et al, 1997; Hong et al, 2005; Courtney et al, 2006;).

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A integridade do DNA é vital para a divisão celular e alterações oxidantes podem interferir na transcrição, translação e replicação do DNA e podem aumentar as mutações, senescência e morte celular (Miranda et al 2008; Ribeiro et al. 2008). Devido a sua abundância nas células e suscetibilidade a oxidação, os ácidos graxos poliinsaturados (AGP) são, para os oxidantes, alvos mais prováveis do que o DNA (Wagner et al, 1994; Shimizu et al, 2001). É estimado que aproximadamente 60 moléculas de ácido linoléico e 200 de ácido araquidônico são consumidas por oxidantes que reagem com a bicamada lipídica. Como essa oxidação desencadeia uma cascata autocatalítica que gera numerosas substâncias genotóxicas, tais danos aos lipídeos tem grandes implicações para a integridade do DNA (Wagner et al, 1994). A peroxidação dos lipídeos de membrana inicia quebras autocatalíticas com conseqüente formação de metabólitos citotóxicos e genotóxicos tais como hidroxinomenal e o malonaldeído. A degradação desses produtos pode interferir na cascata de sinalização intracelular envolvendo replicação e morte das células (Eder et al. 2008). Lipídeos dietéticos, relacionados ao ataque pró-oxidativo das células epiteliais do cólon, podem ser um importante fator de contribuição para a carcinogênese (Udilova et al, 2003; Nowak et al, 2007). Até o momento, ainda não existe um tratamento específico para as DIIs, e a melhor estratégia para regular a resposta inflamatória exacerbada é interferir nos múltiplos estágios da cascata inflamatória com drogas antiinflamatórias e imunossupressoras. Essas drogas, entretanto, apresentam sérios efeitos colaterais que limitam o seu uso (Stein et al, 2000). O tratamento dietético pode ser uma

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alternativa para a terapia medicamentosa (Camuesco et al, 2005; Nowak et al, 2007). As DIIs ocasionam deficiências nutricionais, tais como desnutrição calórica e protéica e deficiência de vitaminas, minerais e oligoelementos, que ressaltam a importância da terapia nutricional em seu tratamento (Pizato et al, 2005; Ferguson et al, 2007; Razack et al, 2007). Desnutrição é comum nestes pacientes, e intervenções, através da terapia nutricional adequada para restabelecer o estado nutricional, tem sido associada à melhora do processo de recuperação com melhora do sistema imunológico nos períodos de crises da doença (Razack et al, 2007). Várias características contribuem para a desnutrição observada nos pacientes: (1) Há uma diminuição da ingestão oral de nutrientes associada à dor abdominal e anorexia; (2) A inflamação mucosal associada à diarréia leva à perda de proteínas, minerais, sangue, eletrólitos e elementos traços. Além disso, ressecções múltiplas ou supercrescimento bacteriano no cólon podem ocasionar efeito nutricional adverso como má absorção de micronutrientes; (3) Terapias medicamentosas podem levar à desnutrição. Por exemplo, a sulfassalazina reduz a absorção de ácido fólico, e corticosteróides diminuem a absorção de cálcio e afetam negativamente o metabolismo protéico (Wild et al, 2007). Estudos epidemiológicos têm sido realizados na tentativa de correlacionar fatores nutricionais com DIIs. A composição e conteúdo de lipídeos na dieta têm sido alterados drasticamente nos últimos anos (Wild et al, 2007). Recentes evidências indicam que alterações no tipo e quantidade de ácidos graxos da dieta estão relacionadas a distúrbios metabólicos nas DIIs (Figler et al, 2007). A ingestão de ácido linoléico (18:2 n-6, LA), nos países ocidentais, aumentou drasticamente no século XX, seguida da introdução de margarina e óleos vegetais, o que tem 21

resultado em um aumento significativo da razão AGP w-6:w-3 na dieta (Calder, 2008). A incidência de DIIs é alta nas populações ocidentais e tem aumentado muito nos países em desenvolvimento que tem adotado estilo de vida urbano industrializado com mudanças concomitantes nos hábitos dietéticos, como a maior ingestão de fast food com alto conteúdo de gorduras (Wild et al, 2007). A relação entre resposta inflamatória e dietas enriquecidas com AGP tem sido bastante investigada nos últimos tempos. Muitos estudos têm demonstrado que os AGP podem modificar reações inflamatórias e imunológicas, podendo ser úteis como terapias auxiliares no tratamento de doenças inflamatórias (Kinsella et al, 1990; Serhan et al, 2004). Os AGP da dieta dos tipos w-6 e w-3 são incorporados aos fosfolipídios das membranas celulares podendo influenciar as respostas imunológicas e inflamatórias, por modificarem a fluidez, os sistemas de defesa antioxidantes, e por darem origem a diferentes precursores da síntese de eicosanóides, importantes mediadores inflamatórios (Kinsella et al, 1990; Simopoulos et al, 2003; Calder, 2008). Os AGP w-3, ácido eicosapentaenóico (EPA) e ácido docosahexaenóico (DHA), inibem competitivamente a oxigenação do ácido araquidônico (AA) pela ciclooxigenase, de forma que a ingestão de quantidade elevada de AGP w-3 diminui a capacidade de síntese de eicosanóides derivados do AA, com concomitante aumento na síntese de prostaglandinas (PG) e tromboxanos (TX) da série 3 e leucotrienos (LT) da série 5 (Yaqoob & Calder, 1995) . Por outro lado, o excesso de AGP w-6, em dietas pobres em AGP w-3, pode contribuir para uma superprodução de PGE2, TXA2 e LTB4, potentes mediadores inflamatórios (Kinsella et al, 1990; Simopoulos et al, 2003; Calder, 2008).

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Os eicosanóides produzidos a partir do EPA (AGP w-3) são em geral menos ativos no processo inflamatório do que os eicosanóides derivados do AA (AGP w-6) (Calder, 1996; Calder, 1998; Kikuchi et al, 1998). Estudos clínicos têm mostrado efeitos benéficos da suplementação da dieta com óleo de peixe sobre condições inflamatórias agudas e crônicas (Harbige, 1998; Simopoulos et al, 2002; Innis et al, 2006; MacLean et al, 2005). Tais efeitos têm sido atribuídos à diminuição na geração dos mediadores inflamatórios derivados do AA e produção de mediadores menos potentes derivados do EPA e DHA, após tratamento com dietas enriquecidas com AGP w-3 (Harbige, 1998; James et al, 1998; Zaloga & Marik, 2001). O papel dos AGP w-3 e w-6 no desenvolvimento do câncer tem sido extensivamente estudado em trabalhos epidemiológicos e experimentais. O contrastante papel, na tumorigênese, dos ácidos graxos AGP w-3 como protetores, e dos AGP w-6 como promotores, tem sido uma questão intrigante no campo da nutrição e câncer (Eder et al, 2008). Chung et al, 2003 demonstraram que adutos cíclicos de DNA, provenientes da oxidação de AGP, dependem do tipo de ácido graxo envolvido. Os adutos cíclicos de cadeia curta, como o Acr-, Cro- e Pen-dG, são derivados primariamente dos AGP w-3, enquanto os de cadeia longa, como Hep- e HNE-dG, são formados exclusivamente a partir dos AGP w-6. Além disso, tem sido relatado que os adutos derivados dos AGP w-3, devido à facilidade de ligação, não causam desequilíbrio das bases de DNA, não promovendo lesões mutagênicas (Chung et al, 2003). Estudos têm demonstrado que a suplementação da dieta com óleo de peixe exerce papel protetor na prevenção do câncer de cólon, sugerindo ser essa suplementação

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uma alternativa segura e efetiva ao uso de drogas antiinflamatórias, particularmente aos inibidores da COX-2, drogas que promovem efeitos colaterais importantes, quando utilizados por períodos prolongados (Zhou et al, 2005; Chapkin et al, 2007).

1.1 Justificativa Em nosso laboratório observamos que tanto dietas suplementadas com AGP w-3 como AGP w-6 diminuem a resposta inflamatória aguda, induzida por carragenina. Tal efeito foi parcialmente atribuído aos elevados níveis de corticóides encontrados nesses animais (Silveira et al. 1995). Adicionalmente, verificamos que as dietas promoviam, durante o desenvolvimento do edema de carragenina, alterações similares em mediadores inflamatórios. Ambas as dietas diminuíram a liberação de H2O2 e óxido nítrico (NO) por macrófagos estimulados por carragenina, os níveis plasmáticos de kalicreína (KK) e a liberação de bradicinina (BK) e óxido nítrico (NO) do exsudato inflamatório (Wohlers et al. 2005). Verificamos ainda que essas dietas promoveram, durante o desenvolvimento do edema de carragenina, uma diminuição das citocinas pró-inflamatórias IL-1 e IL-6 tanto no exsudato quanto no soro, e um aumento da citocina antiinflamatória IL-10 (Wohlers et al, 2007). Diante dessas observações e considerando que as drogas antiinflamatórias e imunossupressoras utilizadas para o tratamento das DIIs apresentam sérios efeitos colaterais (Stein et al, 2000), o objetivo deste trabalho foi verificar se dietas normolipídicas ou hiperlipídicas, enriquecidas com AGP w-3 ou w-6, podem contribuir com a diminuição do processo inflamatório do cólon e atenuar o dano oxidativo no DNA dos animais portadores de colite experimental induzida por DSS, diminuindo a possibilidade de mutações genéticas e conseqüentemente o 24

desenvolvimento de neoplasias. A confirmação dessa possibilidade, em modelos experimentais, poderá auxiliar no emprego de estratégias dietéticas, como tratamento complementar das DIIs, que possam ser úteis na diminuição de drogas utilizadas no tratamento, aumento do período de remissão da doença e prevenção do câncer colorretal nos enfermos portadores de colite ulcerativa. Para tal, utilizamos ratos submetidos ao modelo de colite experimental induzida por Sulfato de Sódio Dextran (DSS), que tem sido amplamente utilizado por produzir uma inflamação colônica similar à colite ulcerativa humana. Esse modelo apresenta outras vantagens incluindo simplicidade, alto grau de uniformidade das lesões e infiltração de leucócitos (Egger et al, 2000; Kullmann et al, 2001; Hudert et al, 2006).

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2 – OBJETIVOS

2.1 - Geral Verificar se dietas hiperlipídicas ou normolipídicas, ricas em AGP w-3 ou AGP w-6, alteram o desenvolvimento da resposta inflamatória, a liberação de mediadores inflamatórios e o dano oxidativo do DNA na colite experimental.

2.2 - Específicos Analisar, em ratos submetidos ao modelo de colite induzida por DSS, se dietas hiperlipídicas ou normolipídicas, ricas em AGP w-3 ou AGP w-6 alteram: a) o escore de gravidade da doença b) a incorporação tecidual hepática de ácidos graxos. c) a histopatologia do cólon distal. d) a atividade da enzima mieloperoxidase expressa nos tecidos colônicos. e) a concentração tecidual das citocinas IFNγ, IL-4 e IL-10. f) o nível de dano oxidativo do DNA de células da mucosa colônica g) a concentração plasmática de corticosterona após a indução da colite.

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3 - MATERIAL E MÉTODOS

3.1 - Animais e tratamento O Comitê de Ética em Pesquisa da UNIFESP aprovou todos os procedimentos envolvendo animais (Projeto n0 01588/07). Foram utilizados ratos (rattus norvergicus) da linhagem Wistar, albinos, heterozigotos,

procedentes

do

Centro

de

Desenvolvimento

de

Modelos

Experimentais para Medicina e Biologia (CEDEME), com idade variável entre 28-34 dias e pesando entre 65-70 g para o inicio da alimentação com as dietas experimentais. Os animais receberam água e alimento ad libitum e foram mantidos em temperatura de 24 ± 1 ºC, sob condições de ciclos de luz alternados, claro/escuro, de 12 horas cada. Os animais foram divididos em seis grupos, tendo, durante 47 dias, livre acesso a diferentes tipos de dietas. A colite foi induzida por 7 dias (do 36o ao 42o dia) pela administração de sulfato de sódio dextran (DSS) na água de beber. No 43º dia o DSS foi descontinuado e no 48º dia os animais foram sacrificados, após jejum alimentar de 24 horas. Os grupos experimentais foram divididos de acordo com a composição da ração: 1. Grupo Hiper Soja (HS) - animais alimentados com dieta hiperlipídica (20% de gordura) à base de óleo de soja;

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2. Grupo Hiper Peixe (HP) – animais alimentados com dieta hiperlipídica (20% de gordura) a base de óleo de peixe; 3. Grupo Hiper Soja-Peixe (HSP) - animais alimentados com dieta hiperlipídica (20% de gordura) com 10% de óleo de soja e 10% de óleo de peixe; 4. Grupo Normo Soja (S) - animais alimentados com dieta normolipídica (5%8% de lipídeos) à base de óleo de soja, sendo este grupo utilizado como controle tanto para as dietas normolipídicas quanto para as dietas hiperlipídicas; 5. Grupo Normo Peixe (P) - animais alimentados com dieta normolipídica (5%8% de gordura) à base de óleo de peixe; 6. Grupo Normo Soja-Peixe (SP) – animais alimentados com dieta normolipídica (5% - 8% de gordura) com 2,5 - 4% de óleo de soja e 2,5 – 4% de óleo de peixe.

3.2 - Indução da colite experimental A colite experimental foi induzida a partir do dia 36 até o dia 42 colocando-se DSS a 3%, preparado diariamente, em água ad libitum, para os animais beberem. No dia 43 a suplementação com DSS foi descontinuada e os ratos sacrificados cinco dias após cessar a administração de DSS (dia 48). A partir da indução da colite, observamos diariamente as características das fezes, peso dos animais, ingestão alimentar e consumo de água. Vinte e quatro horas antes do sacrifício os animais foram mantidos em jejum, com água ad libitum.

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3.3 - Preparações das dietas e armazenamento As dietas foram preparadas no laboratório e sua composição definida segundo as recomendações do American Institute of Nutrition (AIN-93) (Reeves et al, 1997). Os animais receberam dieta própria para o crescimento até sessenta dias de idade e dieta para manutenção após esse período. Todos os ingredientes foram pesados e colocados em uma bacia. Água quente foi adicionada gradativamente durante a homogeneização até atingir a consistência adequada para a formação de “pellets”. Os “pellets” foram levados a estufa a 60 ºC por 24 horas. Após o resfriamento à temperatura ambiente, as dietas, devidamente identificadas, foram armazenadas em recipientes plásticos, em temperatura de cerca de 4 ºC e, conforme a demanda dos animais, oferecidas em porções diárias. As seis dietas diferiram somente no tipo de óleo utilizado (óleo de soja, óleo de peixe ou a mistura de óleo de soja e peixe) como pode ser observado na Tabela abaixo.

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Tabela 1. Composição (g/100g) das dietas normolipídicas e hiperlipídicas. Normolipídicas Ingredientes

Soja

Peixe

Soja/Peixe

Caseína

20,0 (14,0)

20,0 (14,0)

20,00 (14,0)

Amido de milho

62,0 (71,1)

62,0 (71,1)

62,00 (71,1)

Celulose

5,0 (5,0)

5,0 (5,0)

5,0 (5,0)

Mineral mix AIN-93

3,5 (3,5)

3,5 (3,5)

3,5 (3,5)

Vitamina mix AIN93

1,0 (1,0)

1,0 (1,0)

1,0 (1,0)

Bitartarato de colina

0,25 (0,25)

0,25 (0,25)

0,25 (0,25)

L-cistina

0,3 (0,18)

0,3 (0,18)

0,3 (0,18)

0,014 (0,007)

0,014 (0,007)

0,014 (0,007)

Óleo de peixe

0

8,0 (5,0)

4,0 (2,5)

Óleo de soja

8,0 (5,0)

0

4,0 (2,5)

Butilhidroquinona

Hiperlipídicas Ingredientes

Hiper Soja

Hiper Peixe

Hiper Soja/Peixe

Caseína

20,0 (14,0)

20,0 (14,0)

20,00 (14,0)

Amido de milho

62,0 (71,1)

62,0 (71,1)

62,00 (71,1)

Celulose

5,0 (5,0)

5,0 (5,0)

5,0 (5,0)

Mineral mix AIN-93

3,5 (3,5)

3,5 (3,5)

3,5 (3,5)

Vitamina mix AIN93

1,0 (1,0)

1,0 (1,0)

1,0 (1,0)

Bitartarato de colina

0,25 (0,25)

0,25 (0,25)

0,25 (0,25)

L-cistina

0,3 (0,18)

0,3 (0,18)

0,3 (0,18)

0,014 (0,007)

0,014 (0,007)

0,014 (0,007)

Óleo de peixe

0

20,0 (20,0)

10,0 (10,0)

Óleo de soja

20,0 (20,0)

0

10,0 (10,0)

Butilhidroquinona

O primeiro número se refere à dieta crescimento (AIN-93-G) e o número entre parênteses se refere à dieta manutenção (AIN-93-M), quando a composição difere do período de crescimento.

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3.4 - Consumo alimentar e peso corporal O consumo das rações foi controlado semanalmente e determinado através da diferença entre a quantidade de ração oferecida para a ingestão e a sobra desta que era pesada no dia seguinte. O peso dos animais foi mensurado semanalmente no mesmo dia e horário, no período antes da indução da colite, e diariamente após o início da indução da colite.

3.5 - Coleta de amostras Treze dias após o início da indução da colite (48º dia de alimentação), os animais foram anestesiados com ketamina e Xilazina (1:1), na dose de 0.1 mL/100g, tricotomizados na região abdominal, e decapitados para retirada das amostras sanguínea. A seguir realizou-se uma incisão abdominal mediana para a remoção de todo o segmento do cólon e reto, que foi pesado e seu comprimento medido, sob carga constante de 2 g. Esse segmento foi aberto longitudinalmente pela borda mesocólica do ceco até a borda anal, sendo lavado em PBS (phosphate-bufferid saline) e subdividido em 2 segmentos: cólon proximal e cólon distal. O cólon proximal foi desprezado e o cólon distal dividido em quatro fragmentos: O primeiro fragmento (2 cm) foi disposto e preso sob superfície plana de isopor com a face mucosa voltada para cima. Em seguida, fixado em solução de formaldeído a 10% e reservado para estudo histopatológico.

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O segundo fragmento (0,5 cm) foi acondicionado em eppendorff contendo protetor de RNAase e conservado em refrigeração à –80 ºC até a realização do “ensaio cometa” para pesquisa de dano oxidativo do DNA. O terceiro fragmento (0,5 cm) foi acondicionado em um frasco seco, congelado imediatamente após a ressecção em freezer a –80 ºC, até a dosagem da atividade da enzima mieloperoxidase. O quarto fragmento (5,0 cm) foi homogeneizado em 4,0 ml de PBS gelado, centrifugado a 1200 r.p.m. por 10 minutos a 4 ºC e o sobrenadante aliquotado e congelado a –80 ºC para posterior dosagem das citocinas por ELISA. O fígado dos animais foi removido inteiro para análise da incorporação tecidual de ácidos graxos.

3.6 - Análise histológica Os fragmentos do cólon distal, destinados ao estudo histológico e fixados em formalaldeído a 10% foram desidratados em sucessivas concentrações crescentes de álcool. A seguir foram submetidos à clarificação em xileno, incluídos em blocos de parafina e submetidos a quatro cortes longitudinais, com 4µm de espessura para montagem das lâminas que foram coradas pela técnica da hematoxilina-eosina. Os parâmetros histológicos foram analisados qualitativamente por patologista experiente em enfermidades do trato digestivo que desconhecia detalhes do segmento a ser estudado.

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A intensidade do processo inflamatório foi classificada em ausente (-), leve (+), moderado (++) e intenso (+++) baseando-se nos seguintes critérios: 0(-) = Normal 1(+) = Leve Polimorfos nucleares na lâmina própria. Criptite ocasional. Mínima destruição glandular e ulceração.

2(++) = Moderada Moderado número de polimorfonucleares na lâmina própria com criptite e abscessos. Alguma destruição glandular.

3(+++)= Intenso Numerosos polimorfonucleares com abundante criptite. Abscesso crípticos. Destruição celular intensa. Ulceração proeminente da mucosa.

3.7 - Escore do Índice de Atividade da Doença (IAD) A partir do início da indução da colite até o sacrifício dos animais (36º ao 47º dia), foram observados diariamente, o peso do animal, a presença de sangue nas 33

fezes e a consistência das fezes. Esses parâmetros foram classificados de acordo com o Escore proposto por Cooper et al (1993) (Tabela 2), utilizado para calcular a média diária do Índice de Atividade da Doença (IAD) para cada animal.

Tabela 2. Escore do Índice de Atividade da Doença (IAD) IAD

Perda de peso

Consistência das

Sangramento

fezes

retal

Normal

Normal

% 0

0

1

1–5

2

5 – 10

3

10 – 20

4

>20

Perda de consistência

Diarréia

Sangramento

O IAD representa índices combinados de perda de peso, consistência das fezes e perda de consistência das fezes, adaptado por Cooper et al. (1993)

3.8 – Atividade da enzima mieloperoxidase (MPO) A determinação desta enzima foi realizada homogeneizando-se, por 15 a 20 segundos, 50 mg do cólon e 1 ml de uma solução de Brometo de Hexadecil trimetilamônio (HTAB hexadecyltrimethylammonium bromide) (5 g de HTAB para 1 litro de tampão Fosfato de Potássio), em gelo. A seguir, a amostra foi centrifugada por 10 minutos, a 4 ºC, e o sobrenadante separado e colocado em placa de 96 poços, mantendo-se a placa em gelo. Em cada poço foi colocado 80 µl do

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sobrenadante e 200 µl de o-dianisidine, sendo a placa imediatamente lida em 460 nm, de 30 em 30 segundos, por 3 minutos.

3.9 Concentrações de citocinas no tecido colônico As concentrações das citocinas IFNγ, IL-4 e IL-10 foram determinadas por ELISA nos fragmentos de cólon homogeneizados, utilizando-se kits de citocinas para ratos R&D System (Minneapolis, USA) Os protocolos dos kits foram seguidos. Primeiramente, placas para ELISA (Nunc-Maxisorb) foram adsorvidas com anticorpos de captura anti-rato em um tampão de adsorção e incubadas a temperatura ambiente durante a noite. No dia seguinte as placas foram lavadas cinco vezes com cerca de 300 ml/well de Tampão Fosfato-Salina (0,01 M), pH 7,3 e 0,05% de Tween 20 (PBS-T). As placas foram incubadas com 300 µl de tampão de bloqueio [PBS (0,01 M, pH 7,3) e 2% de albumina bovina sérica] durante 2 horas à temperatura ambiente. Após a remoção da solução de bloqueio (lavagem por 5 vezes das placas com PBST), as amostras ou padrões apropriadamente diluídos (volume final = 100 ml) foram adicionados, em duplicata, às placas e incubados por 2 horas. Após um novo ciclo de lavagens, o segundo anticorpo de detecção biotinilado foi adicionado e incubado por 2 horas à temperatura ambiente. Após as lavagens, 100 ml do reagente de detecção (streptoavidina conjugada com peroxidase, na proporção de 1:200) foram adicionados e incubados por 1 hora à temperatura ambiente, em local protegido da luz. Após nova lavagem, foram adicionados 100 ml de substrato TMB (3, 3´, 5, 5´ tetrametilbenzinadina). As placas foram novamente incubadas por 1 hora à

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temperatura ambiente, em local escuro. A reação foi interrompida pela adição de 50ml de H2SO4 (1M) e a intensidade da cor amarela (densidade óptica) determinada em leitor de microplaca (Labsystems Multiskam, MS) a 405 nm. Elaborou-se uma curva padrão de concentrações conhecidas de citocinas e com base nesta curva os resultados foram calculados e expressos em ρg de citocinas por ml de homogeneizado.

3.10 - ENSAIO COMETA O ensaio do Cometa foi realizado no Laboratório de Biologia Molecular da Unidade de Farmacologia e Gastroenterologia (UNIFAG) da Universidade São Francisco, Bragança Paulista. Através deste foi possível detectar danos no DNA em células colônicas. Para tal usamos 0,5 cm do cólon distal. A análise de danos nas células colônicas foi feita de acordo com o Pool-Zobel et al (1994). As amostras foram incubadas em 3 ml de uma solução de Hank's (HBSS, Invitrogen, Carlsbad, CA, USA) contendo 5,5 mg de proteinase K (Sigma Chemical CO., St. Louis, MO) e 3 mg de colagenase I (Invitrogen) por 45 minutos a 37 ºC para a liberação das células. Estas, então, foram ressuspendidas em 10 ml de HBSS e centrifugadas para o isolamento das células. Alíquotas foram retiradas e a viabilidade celular avaliada. A versão alcalina do ensaio do cometa foi realizada de acordo com Ladeira et al (2004). Em suma, 15 ml da suspensão celular previamente obtida foram misturados a agarose low melting point 0,5 % (Promega), postos sobre uma lâmina e cobertos com uma lamínula. Estas foram imersas em uma solução de lise gelada (2,5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% SDS, pH 10 com 1% Triton X-100 e DMSO) e permaneceram a 4 ºC 36

overnight. Subseqüentemente, expostas a um tampão alcalino (1 mM EDTA e 300 mM NaOH, pH~13,4) por 40 min a 4 ºC. A eletroforese foi realizada neste tampão a 4 ºC por 30 min a 25V e 300 mA. Após a corrida, as lâminas foram neutralizadas (0,4 M Tris, pH 7,5), coradas com SYBR Safeä (Invitrogen) e analisadas com um microscópio de fluorescência. Duzentas células foram aleatoriamente selecionadas (100 de cada réplica) e analisadas usando o software Komet 5,5 (Kinetic Imaging, USA). As amostras foram avaliadas e a média do Olive Tail Moment DNA foi determinada. Uma maior porcentagem de DNA na cauda significa altos níveis de danos ao DNA.

3.11 - Composição de ácidos graxos das dietas e incorporação no fígado Para a extração total de lipídeos, as amostras de tecidos congeladas ou de dieta, foram homogeneizadas em clorofórmio e metanol (2:1 v/v) seguida de adição de solução aquosa de KCL (Folch et al. 1957). O clorofórmio foi seco em camadas sob N2 e a extração total foi convertida dentro de ésteres metil de ácidos graxos usando BF3 metanol, de acordo com o método sugerido pelo the American Oil Chemist's Society (1993). Os ésteres metal foram diluídos em hexane e analisados por cromatografia gasosa usando cromatográfico CHROMPACK® chromatographer (model CP 9001) com o detector de ionização e a coluna capilar CP-Sil 88 (Chrompak, WCOT Fused Silica 59 m x 0.25 mm). O detector de temperatura foi 280 o

C e o injector 250oC. A temperatura inicial foi 180 ºC por 2 minutos, programado

para aumentar 10 ºC por minuto até 210 ºC por 30 minutos. O carregador a gás usado foi hidrogênio na freqüência de fluxo de 2,0 mL/ minuto. A identificação dos ácidos graxos foi comparada ao tempo de retenção dos componentes das amostras

37

com um autêntico padrão de ésteres de ácidos graxos injetados sob as mesmas condições. A composição dos ésteres de ácidos graxos, como porcentagem do peso total de ácidos de graxos, foi calculada usando contagem da área do cromatógrafo.

3.12 – Determinação de Corticosterona Plasmática A concentração plasmática de corticosterona foi determinada pelo método fluorimétrico

de

Guilhemim

et

al

(1959)

baseado

na

fluorescência

dos

glicocorticóides em ácido sulfúrico. Uma quantidade de 500 µl de plasma era misturada a 6 ml de diclorometano. Após agitação, formava-se um sobrenadante turvo que era aspirado, usando-se uma pipeta Pauster conectada à bomba de vácuo (Bomba de vácuo e compressor rotativo Primar – Mod. 141, tipo 2 VC). Em seguida, a amostra era submetida à agitação e aspiração após ser acrescida de 0,5 ml de água destilada. Então, 1 ml de uma solução formada por etanol e ácido sulfúrico (7:3) foi adicionada, e a amostra, após agitação, era deixada em repouso até que a solução etanol-sulfúrico, contendo o material fluorescente, precipitasse completamente no fundo do tubo (20 a 30 minutos). Essa solução era cuidadosamente retirada e colocada em cubetas para leitura em espectrofotoflurímetro (Perkin-Elmer LS 5B). Juntamente com as dosagens das amostras foi feita uma curva padrão de três pontos em duplicata, com concentrações conhecidas de corticosterona. Foi calculada a reta de regressão, em função da qual, a fluorescência das amostras foi transformada, e o resultado expresso em microgramas de corticosterona por decilitro de plasma (µg/dl).

38

3.13 – Análise estatística A análise estatística foi realizada com o auxílio do programa PRISMA 4.0 para Windows. Calculou-se o coeficiente de variabilidade (CV) das amostras para a escolha da análise estatística Paramétrica (CV < 0,20), ou Não Paramétrica (CV > 0,20). Como alguns grupos apresentaram um CV maior do que 0,20 (20%), utilizamos estatística Não Paramétrica. Para as comparações feitas entre os grupos utilizou-se a Análise de Variância de Bonferroni. Os dados foram expressos como média ± erro padrão da média (e.p.m) e o nível mínimo de significância foi fixado em 5% (p<0,05). Para a verificação da correlação entre duas variáveis, foi utilizado o coeficiente de correlação de Pearson (r).

39

4 – RESULTADOS E DISCUSSÃO

Os resultados e a discussão estão apresentados no formato de 2 artigos científicos:

- Artigo 1: Soybean and Fish oil mixture increases IL-10, protects against DNA damage and decreases colonic inflammation in rats with dextran sulfate sodium (DSS) colitis

- Artigo 2: High-fat diets enriched with fish or soybean oil do not exacerbate dextran sulfate sodium (DSS)-induced colitis and the soybean/fish oils mixture exerts beneficial effects on cytokines balance and reduces DNA damage

40

Artigo 1: Soybean and Fish oil mixture increases IL-10, protects against DNA damage and decreases colonic inflammation in rats with dextran sulfate sodium (DSS) colitis

Karina Vieira de Barros, MD,1 Roberta Araujo Navarro Xavier, MD,1 Gilclay Gomes de Abreu1 Carlos Augusto Real Martinez, PhD,2 Marcelo Lima Ribeiro, PhD,3 Alessandra Gambero, PhD,3 Patrícia de Oliveira Carvalho, PhD,2 Claudia Maria Oller do Nascimento, PhD,1 Vera Lúcia Flor Silveira, PhD,4.

1

Department of Physiology, Federal University of São Paulo (UNIFESP), São Paulo,

SP, Brazil;

2

Multidisciplinary Research Unit, São Francisco University Medical

School, Bragança Paulista, SP, Brazil;3 Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP, Brazil; 4

Department of Biological Sciences, Federal University of São Paulo, Diadema, SP,

Brazil.

Corresponding author Prof. Dr. Vera L. F. Silveira Departamento de Ciências Biológicas Universidade Federal de São Paulo - Campus Diadema Rua Prof. Artur Riedel, 275, Bairro Eldorado – Diadema – SP – Brasil, CEP: 09972270, Tel./fax: 55-(11)-5576-4527 E-mail: [email protected] / [email protected]

Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

41

ABSTRACT Background: Ulcerative colitis (UC) is characterized by recurrent episodes of colonic inflammation with an imbalance in the synthesis and release of cytokines. Chronic colonic inflammation has been associated with DNA damage and colorectal cancer. Dietary polyunsaturated fatty acids (PUFA), can modulate immune function, inflammation and carcinogenesis. Therefore, we investigated whether PUFA could influence colonic injury, plasma corticosterone and tissue myeloperoxidase activity (MPO), DNA damage and cytokines in colitic rats. Methods: Male weaning Wistar rats were fed for 47 days with an AIN-93 diet with control (C), fish (F) or a mixture of fish and soybean oil (SF). UC was induced from day 36 until day 42 by 3% DSS in drinking water. On day 48, the rats were anesthetized and, blood samples were collected for corticosterone determination. The distal colon was excised for histological analysis and to quantify the cytokine (IL4, IL-10 and INF-γ), MPO and DNA damage. The disease activity index (DAI) was recorded daily during colitis induction. Results: DAI, MPO, histological analyses showed decreases (p<0.05) only in the SF group compared with the C group. IL-10 was increased and DNA damage was reduced in the groups F and SF, and an inverse correlation between these variables (r=0.77) was found. There were no differences in corticosterone, IFN-γ and IL-4 levels. Conclusions: Soybean and fish oil mixture may be effective in improving colonic injury and DNA damage, and it could be an important complementary therapy in UC to reduce the use of anti-inflammatory drugs and prevent colorectal cancer.

Running head: PUFA and colonic inflammation

Key words: Polyunsaturated Fatty Acids, inflammatory bowel disease, DNA damage and Cytokines

42

INTRODUCTION

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by recurrent episodes of colonic inflammation and tissue regeneration1. Although the pathogenesis of UC has not been entirely elucidated, the chronic relapsing inflammation has a multifactorial etiology. UC can be caused by an exaggerated immune response to the intestinal flora in the context of genetic predisposition2,3 that can be attributed, at least in part, to an imbalance between effector T cells (Teff) and regulatory T cells (Treg)4. In IBD, there is increased synthesis and release of pro-inflammatory mediators, such as eicosanoids, platelet activating factor, reactive oxygen species (ROS), nitrogen metabolites, chemokines and mainly cytokines5 that have been associated with disease severity, activity and remission2. Active episodes of UC are characterized by mucosal injury, increased vascular permeability, infiltration of neutrophilic polymorphonuclear leukocytes, disruption of extracellular matrix and epithelial cell damage where neutrophils can mediate cell and tissues injury by the synthesis and release of ROS6. Patients with IBD are at increased risk of developing colorectal cancer, and the inflammation has been associated with neoplastic changes through production of pro-inflammatory cytokines and ROS7. Both mediators activate nuclear transcription factor-kB (NF-kB), inducible nitric oxide synthesis, and cyclooxygenase-2-related signaling pathways, which may retard or suppress apoptosis in intestinal epithelial cells and modulate angiogenesis8. ROS, which are the cellular consequences of oxidative stress, may cause DNA oxidation resulting in damage to all four bases and

43

the deoxy-ribose-molecule9,10. Chronic inflammation in the colonic mucosa caused by increased and continuous exposure of ROS promotes oxidative DNA damage of the epithelial cells, triggering the appearance of genetic mutations and initiating colorectal carcinogenesis10,11. Disturbances of fatty acid status are related to IBD12, and nutrition and dietary factors, mainly fatty acids, can modulate immune function. Dietary fatty acids such as omega 3 (w-3) polyunsaturated fatty acids (PUFA) can exert an anti-inflammatory effect reducing pro-inflammatory cytokines production4. The main purpose of this study was to examine the effect of diets enriched with fish oil, soybean oil and fish plus soybean oil mixture on markers of colonic injury, cytokines (IL-4, IL-10 and IFN), MPO activity, corticosterone levels and DNA damage in colon of rats with experimental UC induced by dextran sulfate sodium (DSS). Colitis induced by DSS is widely used due to the advantages of simplicity, degree of lesion uniformity, and leukocytes infiltration13. DSS is also an experimental model for oxidative stress11,14.

MATERIALS AND METHODS

Animals and diet treatments: Eighteen male Wistar rats (28-30 days) were obtained from the Center for the Development of Experimental Models in Medicine and Biology at the Federal University of São Paulo. They were kept under controlled light conditions (12:12 h light-dark cycle with lights on at 07:00 A.M.) and temperature conditions (24 ± 1°C) with free access to food and water. The animals were separated into three groups

44

(n=6 per group) and received, for 47 days, one of three diets: control (C group), fish (F group) or soybean-fish (SF group) diet. All of the experiments reported were previously reviewed and approved by Institutional Ethics Committee for Experimental Research. The diets were prepared according to the recommendations of the American Institute of Nutrition. The standard AIN-9315 G (8% fat until 2 month) and M (5% fat after 2 month) diets contained the same amount of protein, carbohydrates and lipids. The only difference between the diets was the source of lipids: 100% of soybean oil (source of w-6 PUFA) in the C group, 100% of fish oil (source of w-3 PUFA) in the F group and a mixture of 50% of soybean oil and 50% of fish oil in the SF group. We obtained soybean oil and fish oil from Brazilian producers. The detailed compositions of the diets are presented in Table 1, and the fatty acid profile of each diet is presented in Table 2.

Induction of colitis, samples collection and procedures Colitis was induced in all animals from day 36 to day 42 with 3% DSS (wt/v, prepared daily, mol wt 5.000-Fluka BioChemika) put in the drinking water. Animal body weight, presence of gross blood in the feces and stool consistency were recorded daily for each rat from day 35 to day 47. These parameters were each assigned a score according to the criteria proposed by Cooper et al.16, which was used to calculate a daily mean disease activity index (DAI). Food and water consumption was also recorded daily during this period. On day 47, the rats, which were food deprived for 24 h, were anesthetized (1:1 xilazine-ketamine). Blood samples were collected by decapitation for plasma

45

corticosterone determination (trisodium citrate, as anticoagulant). The distal colon was immediately excised, rinsed with phosphate buffered saline (PBS), weighed, and its length was measured under a constant load (2 g). The distal colon was longitudinally opened and subsequently divided into four segments: 2 cm to histological analysis (immediately fixed in 10% formaldehyde), 0.5 cm to DNA damage detection (maintained in a fixative solution described below), 0.5 cm to myeloperoxidase (MPO) activity determination, 0.5 cm to fatty acids composition and 6.0 cm to cytokines (IL-4, IL-10 and INF-γ) measurements. All samples were stored at -80 °C, except the histological analysis samples .

Histological analysis A cross-section of the distal colon (2 cm) was fixed in 10% paraformaldehyde solution. Afterwards, it was cut into small fragments, dehydrated through an ethanol series (70%–100%), cleared in xylol and embedded in paraffin. The fragments were sliced into 5 µm thick sections and stained with hematoxylin-eosin. Histological evaluation was done by a pathologist who was blinded to the experimental groups, and it was based on the intensity of mononuclear and polymorphonuclear infiltrates in the lamina propria, crypt dilation, cellular destruction and mucosal ulceration. Histopathological changes were graded according to the degree of inflammation using the following scale: absent (0), light (1), moderate (2) and intense (3), and the numbers represented the inflammation score (IS). Results were expressed as mean values of IS ± standard error of the mean (SEM) for each experimental group.

46

Fatty acids composition of diets For total lipid extraction, diets samples were homogenized in chloroform and methanol (2:1 v/v) followed by the addition of an aqueous solution of KCL17. The chloroform layer was dried under N2, and the total extract was converted into methyl esters of fatty acids using BF3 methanol, according to the method suggested by the American Oil Chemist's Society18. The methyl esters were diluted in hexane and analyzed by gas chromatography using a CHROMPACK® chromatographer (model CP 9001) with a flame ionization detector and a CP-Sil 88 capillary column (Chrompak, WCOT Fused Silica 59 mm x 0.25 mm). The detector temperature was 280 oC, and the injector temperature was 250 oC. The initial temperature was 180 oC for 2 minutes (min), programmed to increase 10 oC per min up to 210 oC and held for 30 min. The carrier gas used was hydrogen at a flow rate of 2.0 mL.min-1. The identification of the fatty acids was done comparing the retention times of the sample components with authentic standards of fatty acid esters injected under the same conditions. Fatty acid composition, as a percent of total acid weight, was calculated using area counts of the chromatogram.

Measurement of colon cytokine and plasma concentration Tissue samples were homogenized in 3.5 mL PBS solution and centrifuged at 1200 rpm for 10 minutes. Supernatants were transferred into clean Eppendorf tubes and stored at –80 ºC. The concentration of INF-γ, IL-4 and IL-10 were measured by enzyme-linked immunosorbent assay technique using commercially available kits purchased from R&D Systems. The plasma corticosterone concentration was quantified by the fluorimetric method19. 47

Myeloperoxidase Activity in the Colon Tissues colon samples (0.5 cm) obtained from the distal colon were homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. For the myeloperoxidase (MPO) assay, 50 µL of each sample were added to 200 µL of o-dianisidine solution (0.167 mg/mL odianisidine dihydrochloride, 0.0005% hydrogen peroxide in 50 mM phosphate buffer, pH 6.0) immediately prior to reading the change in absorbance at 460 nm over 5 minutes using a microplate reader (Multiscan MS, Labsystems, Helsinki, Finland).

Comet Assay The Comet assay detects DNA damage (strand breaks and alkali-labile sites) at the individual cell level. Cells from distal colon samples were isolated as described below. The biopsies were pooled and incubated with 5.5 mg proteinase K (SigmaAldrich, St. Louis, MO, USA) and 3 mg collagenase (Invitrogen Life Technologies, Grand Island, NY, USA) in 3 mL of Hank's balanced salt solution (HBSS; Invitrogen) for 45 min at 37 °C to liberate the cells; the cell s were then re-suspended in 10 mL of HBSS. The resulting suspensions were centrifuged at 750 g for 5 min, and the supernatant was discarded. Since high leukocyte content could lead to a bias in the levels of DNA damage from colon cells, leukocyte contamination was assessed in the cell suspensions. Aliquots of 100 mL were dropped onto a slide, fixed with acetone, and stained with hematoxylin and eosin. The slides were analyzed by blinded examiners for leukocyte levels.

48

Cell Viability: The Comet assay should be performed only on samples having a cell viability of more than 75%. Therefore, cell viability was determined using the fluoresceindiacetate (FDA)/ethidium bromide (EtBr; Sigma-Aldrich, St. Louis, MO, USA) assay. Briefly, a fresh staining solution was prepared containing 30 mL FDA in acetone (5 mg/mL), 200 mL EtBr in PBS (200 mg/mL), and 4.8 mL PBS (Invitrogen). The single cell suspension (25 mL) was then mixed with 25 mL of the staining solution, spread onto a slide and covered with a coverslip. Viable cells appeared fluorescent-green, whereas red-stained nuclei indicated dead cells. At least 200 cells were counted per sample.

Determination of DNA Damage: The alkaline Comet assay was performed on the single cell suspensions, according to Singh et al.20, but with some modifications. Briefly, 15 mL of the single cell suspension (~ 2 x 104 cells) were mixed with molten 0.5% low-melting-point agarose (Promega Co. Madison, WI, USA) and spread on agarose-precoated microscope slides. The slides were immersed overnight at 4 °C in freshly prepared cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 2% sodium salt NLauryl sarcosine, pH 10, with 1% Triton X-100 and 10% DMSO; all from SigmaAldrich). Subsequently, the cells were exposed to alkaline buffer (1 mM EDTA and 300 mM NaOH, pH ~13.4) at 4 °C for 40 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was then conducted in the same solution at 4 °C for 20 min using 25 V and 300 mA. After incubation, the slides were washed in cold PBS; lysis, denaturation and electrophoresis were then performed in the same manner as described above. After electrophoresis, the slides were neutralized (0.4 49

M Tris, pH 7.5), stained with 40 mL EtBr (20 mg/mL) and analyzed with a fluorescence microscope (Eclipse E400; Nikon, Melville, NY, USA), using an image analysis system (Komet 5.5; Kinetic Imaging, Nottingham, UK). Two hundred randomly selected cells (100 from each of two replicate slides) were evaluated from each sample, and the mean of the Olive tail moment DNA was determined. Tail moment (TM) is defined as the product of DNA in the tail, and the mean distance of migration in the tail is calculated by multiplying tail intensity/sum comet intensity by the center of gravity of the tail – peak position. A higher percentage of tail DNA signifies a higher level of DNA damage.

Statistical analysis Data were expressed as means ± standard error of the mean (SEM). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Bonferroni’s post hoc test for multiple comparisons. Significance level was set at p< 0.05. To verify the correlation between two variables, Pearson’s correlation coefficients (r) were used.

RESULTS

Fatty acids composition of diets Table 2 shows the fatty acid composition of diets. The levels of monounsaturated fatty acids (MUFA) were not different among the diets. The saturated fatty acid (SAFA) was higher in the F group as compared with the C and SF groups. In the SF diet, in which fish oil was mixed with soybean oil, there was no

50

excess of SAFA like there was in the Fish diet. In relation to PUFA, the F group had a lower amount compared with the C and SF groups; however, the main difference among the diets was the w-6:w-3 PUFA ratio. The C group showed a w6:w3 ratio that was similar to western diets (10:1) while the F group had a ratio (1:6) with an excess of w-3 PUFA. In the SF group, a balanced ratio (2:1) was observed.

Body weight before and after colitis induction, disease activity index (DAI), colon length, inflammation score (IS) and corticosterone. Animals were monitored from weaning, and their weight was evaluated once a week before induction and daily after colitis induction. Up to day 36, there was no difference in weight among the groups; however, after colitis induction, groups SF and F presented with body weight increases compared with the C group (Table 3). The induction of colitis resulted in significant changes in body weight, stool consistence, fecal blood, food intake and a worsened general status. The three groups presented a progressive DAI from the 3rd day with DSS (day 38) until the 7th day (day 42) (Figure 1). During colitis induction, the F group tended to be similar to the SF group in stool consistency and weight loss; however, rectal bleeding was more intense (data not shown). After stopping DSS (day 43), the clinical symptoms showed a regression, normalizing in day 47. The animals were sacrificed at day 48 (recuperation phase) without any clinical symptoms (Figure 1), although the colon damage remained as the histological examination graded by a pathologist (Figure 2).

51

SF group presented a significantly decreased DAI on days 41, 42 and 43 when compared with the C group. The F group had an intermediate DAI, which was between those for the C and SF groups (Figure 1). Colon length, an inflammation indirect marker, was higher in the SF and F groups in relation to the C group (Table 4). In addition, IS revealed typical inflammatory changes in the colonic architecture (ulceration, crypt dilation, mixed cell infiltration and granulocytes) in all groups, but only the SF group presented with lower tissue damage when compared with C (Figure 2), probably associated with a decreased incidence of diarrhea, blood in feces and smaller weight loss in this group. Regarding plasma corticosterone levels, there were no differences among the groups.

Cytokines, MPO activity and DNA damage MPO activity was significantly lower in the SF group than in the C group, suggesting a reduced neutrophil infiltration in colon tissues, and again, the F group had an intermediate result (Table 5). Interestingly, no difference was observed in the IL-4 and INF-γ cytokine tissue concentrations. However, in relation to IL-10, an important cytokine in maintaining gastrointestinal mucosal homeostasis, increased values were found in the F and SF groups when compared with the C group (Table 5). The detected DNA damage levels in colon were significantly lower in the SF and F groups (Table 5), suggesting an inverse association between decreased DNA damage and increased IL-10 levels. In fact, a linear correlation test between these variables was performed and a strong association between an increase in IL-10 and decrease in DNA damage (r= 0.77) was found (Figure 3). 52

DISCUSSION The results showed that the colon inflammation induced by DSS was significantly less severe in group SF, showing that the mixture of fish oil and soybean oil balanced the w6:w3 PUFA ratio (2:1) and improved colonic inflammation. The diet enriched with fish oil (F group) presented intermediate effects, probably because of the imbalance in the w-6:w-3 PUFA ratio (1:6). Several studies demonstrated the importance of modulating the w-6: w-3 ratio to obtain beneficial effects rather than simply reducing w-6 PUFA levels21. The imbalance in the w-6:w-3 ratio, as is observed in western diets (10:1), may be related to an increased production of proinflammatory cytokines and eicosanoids in autoimmune diseases and IBDs22. Fish oil contains large amounts of SAFA, which has been associated with chronic diseases23,24. Several studies indicate that the optimal w-6:w-3 ratio may vary according to the disease; however, the ratio between 5-2:1 has been associated with decreased inflammation in patients with IBD, rheumatoid arthritis and other inflammatory diseases and with reduced rectal cell proliferation in patients with colorectal cancer25. It has been suggested that patients with IBD show changes in the metabolism of long chain polyunsaturated fatty acids. These alterations may be relevant in maintaining the chronic inflammatory activity in the colon26. Fish oil supplementation may be able to down-regulate the expressions of some genes, which have been involved in inflammatory process12,27. In this paper, we evaluated animals in the recuperation phase, 5 days after stopping DSS exposure. We decided to evaluate this period for considering diet as a complementary therapy; in a severe inflammation model, the benefit effects would be

53

more difficult to observe. The protocol was started using DSS 5% in drinking water for 7 days, and later, it was decreased to 2% for 10 days, when the animals were sacrificed21. However, the mortality in this experimental model was 80%, and so we decided to decrease the DSS to 3% for 7 days and water for 5 days. This high mortality could be associated with different molecular weight of DSS and/or the animals’ gender. The mol wt of 36,000 – 50,000 for DSS and Wistar female rats were not used in this paper. Intense rectal bleeding observed in the F group during colitis induction could be associated with a decreased production of thromboxanes A2, a potent platelet aggregator, as it has been demonstrated with fish oil rich-diets23,25,28. The DAI evaluation showed lower values in the SF group suggesting that the balance of fish and soybean oils exerts protective effects in decreasing disease activity and protecting against weight loss. These are important factors considering that the nutritional status of patients with IBD is negatively affected during the disease activity and that dietary interventions could be beneficial and improve clinical and nutritional status29. In this paper, increased IL-10 levels in the SF and F groups could be associated with a protective effect of diets on weight loss, colon length and DNA damage, important inflammatory factors in IBD. Cytokines play a key role in the development, recurrence and exacerbation of the inflammatory process in IBD. IL-10 is an immunoregulatory cytokine that influences the immunological system, both on the innate and cell-mediated response. It affects the gastrointestinal mucosal homeostasis through the down-regulation of colon inflammation and the inhibition of both antigen presentation and release of pro-inflammatory cytokines, and it is related to the activity of regulatory cells 2,30,31.

54

No difference was found in INF-y and IL-4 cytokines. Evaluating cytokine release in experimental colitis, Dieleman et al.32 also found no increase in IL-4 and INF-γ in the acute phase of UC. These authors observed these elevated cytokines only in later phases (14 days after DSS stopping). An

increase

in

the

colonic

MPO

activity,

a

specific

marker

of

polymorphonuclear neutrophils activity, was used as a measure of the inflammatory status33. The present study showed that the MPO activity decreased only in the SF group in relation to the C group, which matched the lower inflammation based on the inflammatory score and decreased DNA damage. Once more, the balanced w6:w3 ratio used in the SF group shows a beneficial effect on UC. In fact, neutrophils may mediate a mucosal injury by the synthesis and release of ROS6, and it has been known that oxidative stress is a pathogenic factor correlated with DNA damage9. There was a clear correlation between increased IL-10 levels and decreased DNA damage, and to the best of our knowledge, this is the first demonstration of a causal association between these variables associated with IBD. It is likely that the anti-inflammatory effects from IL-10 attenuating mucosal inflammation are associated with putative protection in DNA. Considering that inflammation can accelerate tumorigenesis in the colon, and anti-inflammatory drugs have been used to prevent this event, a dietary intervention with a mixture of fish and soybean oil may be an effective complementary therapy to prevent cancer. Also, it could be an alternative to the use of anti-inflammatory drugs and their associated side effects. Some studies have concluded that there are some benefits of fish oil in IBD34,35,36. However, a recent study reported no effect of w-3 PUFA on disease activity in humans IBD37. These controversial results have been attributed to the 55

different w-3 PUFA doses used28. Here, the mixture of fish and soybean oil in the diet was demonstrated to be better than the use of fish oil as an exclusive source of fat. The experimental conditions of this study could represent a stressful situation for the rats, and they could modify their corticosterone levels. It has been reported that plasma corticosterone levels are significantly increased when animals undergo a single stress session, compared with chronic stressful conditions38. Furthermore, it has been shown that chronic stress does not modify either catecholamine or corticoids plasma levels, indicating an adaptation of the neuroendocrine circuit39. Here, we show that plasma corticosterone levels did not differ in the three experimental groups. This strongly suggests that the animals had adapted at the time of the experiment and that the anti-inflammatory effects obtained by dietary treatment using fish or soybean/fish diet could not be attributed to the action of this hormone. In conclusion, the main beneficial effects exerted by the balance between fish and soybean oil were in reduced disease activity, improved histological score, increased IL-10 cytokine, decreased MPO and protection against DNA damage. The inverse correlation between IL-10 levels and DNA damage also demonstrated that the w-6:w-3 ratio (2:1) was important in reducing disease activity and colon cancer prevention associated with colitis.

Acknowledgments The authors are indebted to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

56

Table 1 - Composition of the experimental diets according AIN-9315 Diet (g/100 g) Ingredient

Control

Fish

Soybean/Fish

Casein+

20.0 (14.0)

20.0 (14.0)

20.00 (14.0)

Corn starch*

62.0 (71.1)

62.0 (71.1)

62.00 (71.1)

Cellulose*

5.0 (5.0)

5.0 (5.0)

5.0 (5.0)

Mineral mix AIN-93@

3.5 (3.5)

3.5 (3.5)

3.5 (3.5)

Vitamin mix AIN 93@

1.0 (1.0)

1.0 (1.0)

1.0 (1.0)

Choline bitartrate*

0.25 (0.25)

0.25 (0.25)

0.25 (0.25)

L-cystina*

0.3 (0.18)

0.3 (0.18)

0.3 (0.18)

0014 (0.007)

0.014 (0.007)

0.014 (0.007)

0

8.0 (5.0)

4.0 (2.5)

8.0 (5.0)

0

4.0 (2.5)

Butylhydroquinone* Fish oil# Soybean oil&

The first number refers to the growth diet (AIN-93 G), and the number in parentheses refers to the maintenance diet (AIN-93 M). +casein was obtained from Sinth. *L-cystine, cornstarch, butylhydroquinone, cellulose and choline bitartrate were obtained from Viafarma, Brazil. Soybean oil& from Lisa, Brazil and fish oil from Campestre, Brazil. Mineral mix@ and vitamin mix@ AIN-93 were supplied by Rhoster Ind. and Com. LTDA.

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Table 2: Fatty acids composition of the experimental diets Percentage of total fatty acids Fatty acids (%)

Control

Fish

Soybean/Fish

14:0

0.6

8.9

2.3

16:0

10.2

20.8

12.9

18:0

3.2

5.5

3.9

16:1 (w-7)

1.6

8.4

4.3

18:1 (w-9)

20.5

9.8

15.2

18:2 (w-6)

49.4

4.8

35.6

18:3 (w-3)

4.9

0.8

2.4

20:4 (w-6)

Nd

Nd

Nd

20:5 (w-3)

Nd

16.2

8.2

22:6 (w-3)

Nd

14.9

6.4

Ni

9.6

9.9

8.8

Total SAFA

14.0

35.2

19.1

Total MUFA

22.1

18.2

19.5

Total PUFA

54.3

36.7

52.6

w-6 PUFA

49.4

4.8

35.6

w-3 PUFA

4.9

31.9

17.0

w-6:w-3 PUFA

10:1

1:6

2:1

SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Nd, not detected, Ni, Not identified.

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Table 3: Values indicate body weight (g) before and after induction of colitis by DSS Days Before induction of colitis

After induction of colitis # 42 47

7

14

21

28

35

129.83

167.49

209.41

244.96

286.25

265.82

278.39

± 3.13

± 2.19

± 3.50

± 3.26

± 2.58

± 5.88

± 7.81

140.70

172.33

224.53

264.47

301.98

290.36

311.69

± 5.05

± 5.29

± 8.07

± 10.74

± 4.14

± 9.04*

± 5.84*

Soybean/

129.57

168.79

210.01

250.23

289.88

301.58

314.86

Fish

± 4.08

± 3.84

± 4.00

± 4.57

± 5.28

± 2.06*

± 6.60*

Groups Control

Fish

Values represent means ± SEM, n= 6 rats; * Different (p < 0.05) from Control group; # DSS 3% from day 36 until day 42.

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Table 4: Colon length (cm), inflammation score and corticosterone concentration (µg/100mL) in DSS-induced colitis rats fed control, fish or soybean/fish diet.

Colon length

Inflammation Score

Corticosterone

Control

14.26 ± 0.24

2.33 ± 0.33

14.57 ± 1.40

Fish

16.85 ± 0.30*

1.83 ± 0.30

14.63 ± 1.11

Soybean-Fish

17.21 ± 0.28*

1.16 ± 0.16*

16.17 ± 0.89

Groups

Values represent means ± SEM, n= 6; * Different (p < 0.05) from Control group.

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Table 5: IL-4, IL-10 and INF-y concentrations (ρg/mL), MPO activity (U/mg) in the colon and DNA damage levels (tail moment/100 cells isolated from colon) of DSSinduced colitis rats fed Control, Fish or Soybean/Fish diet.

IL-4

IL-10

INF-y

MPO

DNA damage

Control

209.36 ± 47.30

310.01 ± 33.36

206.08 ± 37.27

0.84 ± 0.23

4.66 ± 0.16

Fish

255.66 ± 37.25

539.79 ± 57.14*

234.81 ± 21.04

0.60 ± 0.09

3.46 ± 0.11*

SoybeanFish

272.64 ± 44.22

653.50 ± 61.69*

285.15 ± 43.35

0.21 ± 0.06*

2.94 ± 0.19*

Groups

Values represent means ± SEM, n= 6 rats; * Different (p < 0.05) from Control group.

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Figure 1: Time course of changes in the DAI (combined scores of weight loss, stool consistency and bleeding) in rats fed control, fish or soybean/fish diet, based on Cooper et al. criteria16. Values represent means ± SEM, n = 6 rats; * Different (p < 0.05) from Control group.

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A

B

C

Figure 2: Histology (hematoxylin-eosin, magnification, x 200) of colonic samples taken from Wistar rats receiving 3% DSS for 7 days and water for 5 days. (A) Control group, fed control diet, showed ulceration of epithelial superficies (black arrow), intense inflammatory cellular infiltration (white arrow) and destruction of colonic architecture; (B) Fish group, fed fish diet, showed basal lamina edema and moderate cellular infiltrate (arrow); (C) Soybean/Fish group, fed soybean/fish diet showed light cellular infiltrate (arrow). The soybean/fish diet was more efficient than the fish diet in attenuating morphologic damage and preserving colonic architecture.

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Figure 3: Correlation between DNA damage levels (tail moment/100 cells isolated from colon) and tissue IL-10 concentration (ρg/mL) in all DSS-induced colitis rats.

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7. Hegazi RAF, Saad RS, Mady H, Matarese LE, O’ Keefe S, Kandil HM. Dietary fatty acids modulate chronic colitis, colitis-associated colon neoplasia and COX-2 expression in IL-10 Knockout mice. Nutrition. 2006; 22: 275-282. 8.

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16. Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993;69:238-249. 17. Folch J, Less M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497-509. 18. American Oil Chemists' Society (AOCS). Official methods and recommended practices of the American Oil Chemists' Society. 4th ed. Champaigh, 1993. 19. Guillemin R, Clayton GW, Lipscomb HS, Smith JD. Fluorimetric measurement of rat plasma and adrenal corticosterone concentration. J Lab Clin Med. 1959;53:830-2. 20. Sing N, McCoy M, Tice R and Schneider E. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res.1988;175: 184-191. 21. Camuesco D, Gálvez J, Nieto A, Comalada M, Rodriguez-Cabezas ME, Concha A, Xaus J, Zarzuelo A. Dietary olive oil supplemented with fish oil, rich in EPA and DHA (n-3) polyunsaturated fatty acids, attenuates colonic inflammation in rats with DSS-induced colitis. J Nutr. 2005;135:687-94. 22. Calder PC, Immunomodulation by omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids. 2007;77:327-35. 23. Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr. 2002;21:495-505. 24. Courtney ED, Matthews S, Finlayson C et al. Eicosapentaenoic acid (EPA) reduces crypt cell proliferation and increases apoptosis In normal colonic mucosa in subjects with a history of colorectal adenomas. Int J Colorectal Dis. 2007;22:765-76.

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25. Simopoulos AP. Importance of the ratio of omega-6/omega-3 essential fatty acids: evolutionary aspects. World Rev Nutr Diet. 2003;92:1-22. 26. Kuroki F, Matsumoto T, Aoyagi K, Kanamoto K, Fujishima M. Serum n-3 polyunsaturated fatty acids are depleted in Crohn’s disease. Dig Dis Sci. 1997;42:1137-41. 27. Gil Á. Polyunsaturated fatty acids and inflammatory diseases. Biomed Pharmacother. 2002; 56:388-396. 28.

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34. Roediger WE. The starved colon – diminished mucosal nutrition, diminished absorption, and colitis. Dis Colon Rectum. 1990;33:858-62. 35. Belluzzi A. N-3 fatty acids for the treatment of inflammatory bowel diseases. Proc Nutr Soc. 2002; 61:391-395. 36. Belluzzi A. Polyunsaturated fatty acids and inflammatory bowel diseases. Am J Clin Nutr. 2000;71:339S-342S. 37. Trebble TM, Stroud MA, Wootton AS, et al. High dose fish oil and antioxidants in Crohn´s disease and the response of bone turnover: A randomized controlled trial, Br J Nutr. 2005;94:253-261. 38. Dhabar FS, Mcewen BS, Spencer RL. Adaptation on prolonged of repeated stress-comparison between rat strains showing intrinsic intrinsic differences in reactivity to acute stress. Neuroendocrinology 1997;65:360–368. 39. De Boer SF, Koopmans SJ, Slangen JL, Van der Gugten J. Plasma catecholamine, corticosterone and glucose responses to repeated stress in rats: Effects of interstressor interval length. Physiol Behav. 1990;47:1117–1124.

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Artigo 2: High-fat diets enriched with fish or soybean oil do not exacerbate dextran sulfate sodium (DSS)-induced colitis and the soybean/fish oils mixture exerts beneficial effects on cytokines balance and reduces DNA damage

Authors: Karina V. Barros 1, Roberta A.. N Xavier 1, Gilclay G. Abreu 1, Carlos A. R. Martinez 2, Marcelo L. Ribeiro 3, Alessandra Gambero 3, Patrícia º Carvalho 2, Vera L. F. Silveira 4. Department and Institutional addresses: Filiation: 1Department of Physiology, Federal University of São Paulo, São Paulo, SP, Brazil; 2Multidisciplinary Research Unit, São Francisco University Medical School, Bragança Paulista, SP, Brazil; 3

Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical

School, Bragança Paulista, SP, Brazil; 4Department of Biological Sciences, Federal University of São Paulo, Diadema, SP, Brazil.

Running title: Soybean/fish oils alter cytokines and DNA damage

Corresponding author: Prof. Dr. Vera L. F. Silveira Departamento de Ciências Biológicas, Universidade Federal de São Paulo-Campus Diadema. Rua Prof. Artur Riedel, 275, Bairro Eldorado – Diadema – SP, CEP: 09972-270, Brasil, Tel./fax: 55-(11)-5576-4527. E-mail: [email protected] / [email protected]

Word count:4.175

Number of figures: 2

Number of tables: 4

Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) No conflicts of interest

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Abstract

High fat diets have been showed as a risk factor to ulcerative colitis (UC) and colorectal cancer. Omega-6 polyunsaturated fatty acid (PUFA) is considered to increase lipid peroxidation while omega-3 PUFA exerts a chemoprevention. This paper evaluated the effect of high-fat diets (20%) enriched with fish or soybean oil on colonic inflammation and DNA damage in DSS-induced colitis. Male Wistar rats (2830 days) were fed for 47 days with AIN-93 diet divided in 4 groups: control normal fat (5% fat), HS (20% of soybean oil), HF (20% of fish oil) or HFS group (10% of fish plus 10% of soybean oil). UC was induced from day 35 until day 41 by 3% DSS. On day 47 the rats were anesthetized, blood samples were collected for corticosterone determination, and distal colon was excised to quantify IL-4, IL-10 and INF-γ tissue concentration, MPO activity, histological analyses and DNA damage. The disease activity index (DAI) was recorded daily, from day 35 until day 47. The DAI, histological analysis, MPO activity, IL-4, INF-y and corticosterone were not different among the groups. IL-10 was significantly increased by HF diet in relation to HS, but only the SF diet increased IL-10/IL-4 ratio (anti-inflammatory/pro-inflammatory) and reduced DNA damage compared to HS group (p<0,05). The data shows that high fat diets (20%) do not exacerbate the UC and suggests that the soybean and fish oil dietary mixture, more than the only fish oil, could be a complementary therapy in the cytokines balance and colorectal cancer prevention.

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INTRODUCTION

Ulcerative colitis (UC) is a chronic and recidivating inflammatory disease characterized by dysregulation of the immune mucosal response to the intestinal microflora, imbalance in the synthesis and release of cytokines, and an unresolved inflammatory process associated with mucosal damage1,2,3. There is a strong epidemiological evidence linking diet to UC4, where high fat diets are an important environmental risk factor that contributes to the increase incidence of UC in western-style diets5. These diets can cause increased liver inflammation that is associated with depletion of its NK T cells. Moreover they are associated to an imbalance between effectors T cells and regulatory T cells and increased risk to UC6. Intestinal immune system and cytokines play a key role in primary mechanism of pathogenesis in UC. Initially, cytokine profiles are categorized as T helper cells into Th1 and Th2 subsets. Th1 cells produce IL-2 and IFN-γ while Th2 cells produce IL-4, IL-5 and IL-13 which tend to predominate in UC3,7,8. IL-10 is a T regulatory cytokine that influences and contributes to the gastrointestinal mucosal homeostasis and it has been considered as an anti-inflammatory in inflammatory bowel disease (IBD) models9,10,11 Furthermore, IL-4 has been considered as a pro-inflammatory cytokine in IBD models and its colonic overexpression is associated to a severe ulcerative colitis8. Dietary fatty acids, mainly PUFA, are important risk factors in carcinogenesis due to their lipid peroxidation products, by easily oxidation12,13 The tissue high vulnerability to lipid peroxidation has been partly attributed to its high content of long–

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chain polyunsaturated fatty acids, such as arachidonic (PUFA w-6) and docosahexaenoic acids (PUFA w-3), which induce to the formation of reactive oxygen species (ROS) and increase DNA adducts14 The w-6 PUFA has been considered to increase the lipid peroxidation by cyclooxygenases, while w-3 PUFA exerts a chemopreventive role because they suppress lipid peroxidation products formation derived from arachidonic acids oxidation15,16 by competitive inhibition of desaturases. Although the high intake of PUFA has been related to colorectal cancer17. Several studies show that besides the genotoxic effects of lipid peroxidation, epigenetic factors may also be responsible for an increased cancer risk after PUFA excessive intake18. The main purpose of this study was to examine the effect of PUFA-rich diets with fish oil, soybean oil and fish plus soybean oil mixture on markers of colonic injury, cytokines (IL-4, IL-10 and IFN), MPO activity, corticosterone levels and DNA damage in the colon of rats with experimental UC induced by DSS.

MATERIALS AND METHODS

Animals and diet treatments: Male Wistar rats (28-30 days) were obtained from the Center for the Development of Experimental Models in Medicine and Biology at the Federal University of São Paulo. They were kept under controlled light conditions (12:12 h light-dark cycle with lights on at 07:00 A.M.) and temperature conditions (24 ± 1°C) with free access to food and water. The animals were separated into four groups

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(n=6 per group) and received, for 47 days, one of four diets: control normal fat (C group), fish high fat (HF group), soybean high fat (HS group) or soybean-fish high fat (HSF group) diet. All of the experiments reported were previously reviewed and approved by the Institutional Ethics Committee for Experimental Research. The diets were prepared according to the recommendations of the American Institute of Nutrition. The control normal fat group was fed with the standard AIN-9319 G (8% fat until 2 month) and M (5% fat after 2 month) and the high fat groups received 20% fat diet during whole experiment. The high fat diets contained the same amount of protein, carbohydrates and lipids. The only difference between the diets was the source of lipids: 100% of soybean oil (source of w-6 PUFA) in the HS group, 100% of fish oil (source of w-3 PUFA) in the HF group and a mixture of 10% of soybean oil and 10% of fish oil in the HSF group. We obtained soybean oil and fish oil from Brazilian producers. The diets detailed compositions are presented in Table 1, and the fatty acid profile of each diet is presented in Table 2.

Induction of colitis, samples collection and procedures Colitis was induced in all animals from day 36 to day 42 with 3% DSS (wt.v, prepared daily, mol wt 5.000-Fluka BioChemika) put in the drinking water. Animal body weight, presence of gross blood in the feces and stool consistency were recorded daily for each rat from day 35 to day 47. These parameters were each assigned a score according to the criteria proposed by Cooper et al.20, which was used to calculate a daily mean disease activity index (DAI). Food and water consumption was also recorded daily during this period.

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On day 47, the rats, which were food deprived for 24 h, were anesthetized (1:1 v.v xilazine-ketamine). Blood samples were collected by decapitation for plasma corticosterone determination (trisodium citrate, as anticoagulant). The distal colon was immediately excised, rinsed with PBS, weighed, and its length was measured under a constant load (2 g). The distal colon was longitudinally opened and subsequently divided into four segments: 2 cm to histological analysis (immediately fixed in 10% formaldehyde), 0.5 cm to DNA damage detection (maintained in a fixative solution described below), 0.5 cm to MPO activity determination, 0.5 cm to fatty acids composition and 6.0 cm to cytokines (IL-4, IL-10 and INF-γ) measurements. All samples were stored at -80 °C, ex cept the histological analysis samples.

Histological analysis A cross-section of the distal colon (2 cm) was fixed in 10% paraformaldehyde solution. Afterwards, it was cut into small fragments, dehydrated through an ethanol series (70%–100%), cleared in xylol and embedded in paraffin. The fragments were sliced into 5 µm thick sections and stained with hematoxylin-eosin. Histological evaluation was done by a pathologist who was blinded to the experimental groups, and it was based on the intensity of mononuclear and polymorphonuclear infiltrates in the lamina propria, crypt dilation, cellular destruction and mucosal ulceration. Histopathological changes were graded according to the degree of inflammation using the following scale: absent (0), light (1), moderate (2) and intense (3), and the numbers represented the inflammation score (IS). Results were expressed as mean values of IS ± SEM for each experimental group.

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Diet fatty acids composition and liver incorporation For total lipid extraction, diets and tissue samples were homogenized in chloroform and methanol (2:1 v.v) followed by the addition of an aqueous solution of KCL21. The chloroform layer was dried under N2, and the total extract was converted into methyl esters of fatty acids using BF3 methanol, according to the method suggested by the American Oil Chemist's Society22. The methyl esters were diluted in hexane and analyzed by GC using a CHROMPACK® chromatographer (model CP 9001) with a flame ionization detector and a CP-Sil 88 capillary column (Chrompak, WCOT Fused Silica 59 mm x 0.25 mm). The detector temperature was 280 oC, and the injector temperature was 250 oC. The initial temperature was 180 oC for 2 minutes (min), programmed to increase 10 oC per min up to 210 oC and held for 30 min. The carrier gas used was hydrogen at a flow

rate of 2.0 mL.min-1. The

identification of the fatty acids was done comparing the retention times of the sample components with authentic standards of fatty acid esters injected under the same conditions. Fatty acid composition, as a percent of total acid weight, was calculated using area counts of the chromatogram.

Measurement of colon cytokine and corticosterone plasma concentration Tissue samples were homogenized in 3.5 mL PBS solution and centrifuged at 1200 rpm for 10 minutes. Supernatants were transferred into clean Eppendorf tubes and stored at –80 ºC. The concentration of INF-γ, IL-4 and IL-10 were measured by ELISA technique using commercially available kits purchased from R&D Systems. The plasma corticosterone concentration was quantified by the fluorimetric method23.

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Myeloperoxidase Activity in the Colon Tissues colon samples (0.5 cm) obtained from the distal colon were homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. For the MPO assay, 50 µL of each sample were added to 200 µL of o-dianisidine solution (0.167 mg/mL o-dianisidine dihydrochloride, 0.0005% hydrogen peroxide in 50 mM phosphate buffer, pH 6.0) immediately prior to reading the change in absorbance at 460 nm over 5 minutes using a microplate reader (Multiscan MS, Labsystems, Helsinki, Finland).

Comet Assay To carry out the Comet assay, 0,5 cm distal colon was used. Analysis of oxidative DNA damage of the cells in the colonic mucosa was done in accordance with a method previously described24. In summary, the samples were incubated in 3 mL of HBSS (Invitrogen), containing 5.5 mg of proteinase K (Sigma-Aldrich, St. Louis, MO, USA) and 3 mg collagenase (Invitrogen Life Technologies, Grand Island, NY, USA) for 45 min at 37 °C to liberate the cells. The cells were then re-suspended in 10 mL of HBSS and centrifugate to isolate the cells. Aliquots were selected for analysis only if they presented viability > 75%. The alkaline version of the Comet assay was carried out in accordance with Ladeira et al25. In summary, 15 ml volumes of the cell suspension previously obtained were mixed with 0.5% low melting point agarose, placed on slides, and covered with cover slips. Next, the slides were immersed in a cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 2% sodium salt N-Lauryl sarcosine, pH 10, with 1% Triton X-100 and 10% DMSO [dimethylsulfoxide]; all from Sigma-Aldrich) and were left at 4 ºC for 12 hours.

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Following this, the slides were exposed to an alkaline buffer (1 mM EDTA and 300 mM NaOH, pH ~13.4) for 40 minutes at 4ºC. Electrophoresis was carried out in this buffer at 25 V and 300 mA minutes at 4 ºC. After electrophoresis, the slides were neutralized (0.4 M Tris, pH 7.5), stained with 40 mL EtBr (20 mg/mL) and analyzed with a fluorescence microscope (Eclipse E400; Nikon, Melville, NY, USA), using an image analysis system (Komet 5.5; Kinetic Imaging, Nottingham, UK). Two hundred randomly selected cells (100 from each of two replicate slides) were evaluated from each sample, and the mean of the Olive tail moment DNA was determined. Tail moment (TM) is defined as the product of DNA in the tail, and the mean distance of migration in the tail is calculated by multiplying tail intensity/sum comet intensity by the center of gravity of the tail – peak position. A higher percentage of tail DNA signifies a higher level of DNA damage.

Statistical analysis Data was expressed as means ± SEM. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni’s post hoc test for multiple comparisons. Significance level was set at p< 0.05.

RESULTS Fatty acids composition of diets, liver fatty acids incorporation, food intake and body weight As shown in Table 2, there was a higher amount of 18:2 (w-6) and lower 20:5 (w-3) and 22:6 (w-3) in the C and HS groups, fed with soybean oil rich diet, and the

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inverse occurred in the HF and HFS groups fed with fish and soybean oil plus fish oils rich-diets respectively. The total SFA amount was higher in the F group and the total MUFA amount did not differ among the groups. As expected, the different oil sources altered the liver fatty acid composition, as demonstrated in Table 3. There was a higher liver incorporation of arachidonic acid (20:4 w-6) in the C, HS and HSF groups in relation to the HF group, demonstrating that an increased amount of dietary linoleic acid (18:2 (n-6)) was converted in arachidonic acid in the liver tissue. A greater liver incorporation of eicosapentaenoic acid (EPA - 20:5 w-3) and docosahexaenoic acid (DHA - 22:6 w-3) was seen in the HF and FS groups. Food intake and body weight gain were similar among the groups (data not shown).

Disease activity index (DAI), colon length, inflammation score (IS), corticosterone and MPO activity No difference in the DAI was observed among the groups. The disease activity increased progressively from day 36 until to day 42 (during induction of colitis) and decreased from day 43 until day 47 (after DSS stopping). All animals presented changes in body weight, stool consistence, fecal blood, food intake and a worsened general status. (data not shown). Nevertheless, no differences were found in colon length, an inflammation indirect marker, in IS, MPO activity and in corticosterone plasma levels (Table 4).

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Cytokines and DNA damage No difference was observed in IL-4 and INF-γ level. The IL-10 levels were significantly increased only in the HF group in relation to the HS group (Figure 1). In the HSF group an increase of 47% in IL-10 levels was observed as compared with the HS group, but it was not statistically different. Interestingly, when the IL-10/IL-4 ratio (anti-inflammatory/pro-inflammatory cytokines) was performed, just the HFS group had a significant increase as compared with the HS group. Comet assay, a technique used to quantify the oxidative DNA damage in colon cells, demonstrated a decreased DNA damage only in the HSF group when compared with the HS group, showing a protective effect of the HSF diet.

DISCUSSION

Differently of other studies that could find beneficial effects of fish oil in decreasing disease activity in UC, we did not observe differences when fish oil was supplemented in the diets HF and HSF. We observed an increased IL-10/IL-4 ratio associated with a protection against the DNA damage only in the HSF group, when fish and soybean oils were mixtured in the diet. The HF diet increased the immunoregulatory cytokine levels, IL-10, but was not able to protect against DNA damage. We used diets with 20% of total fat without supplemented antioxidants. Bancroft et al26 found a reduction in the accumulation of 8-oxodG, a DNA adducts that is related to the colon cancer, in animals fed with fish oil rich-diet supplemented with 15% of total fat as compared with animals fed with

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corn oil rich-diet. However, they used high quantities of α-tocoferol, gama-tocoferol and t-butylhydroquinone as antioxidant supplementation. It has been known that high fat diets could increase lipid peroxidation, ROS and inflammation worsening. Recently Ma et al6 demonstrated that high fat diets could exert an imbalance in cytokines release and production and to increase susceptibility to DSS-induced colitis. Differing from these authors, which used 50% of total fat, our results did not show worsening in the colitis in all groups receiving diets with 20% of total fat in relation to the C group (5% of total fat). Although dietary fish oil supplementation has been associated with beneficial effects in several inflammatory diseases, some precautions are necessary in relation to the used dose, concerning that these fatty acids are easily oxidated. More studies are necessary to indicate the upper level of these fatty acids to avoid prejudicial effects of lipid peroxidation. As it was demonstrated in Table 3, the liver fatty acidy composition represented the fatty acid given in the diet, showing that PUFA diet content affects the liver fatty acid composition. In fact, this association has been showed also in colon and other tissues27,28. The cytokines play a role in IBD pathogenesis and the IL-10 anti-inflammatory function has been recognized in the intestinal homeostasis. It has been demonstrated that IL-10 knockout mice develop spontaneous chronic colitis and colorectal cancer9,10. Interestingly, pro-inflammatory properties of IL-4 have been demonstrated in the intestinal microenvironment8. Considering this fact, it IL-10/IL-4 ratio was performed and to the best of our knowledge, this is the first demonstration of a causal association between the increase of this ratio and the reduction of DNA damage, that could be observed only in the HSF group in relation to the HS group.

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We have demonstrated in our lab that high fat diet rich in fish or soybean oil could exert beneficial effects on the acute inflammation model. This effect was partially attributed to the basal elevated corticosterone levels induced by these diets29,30 Here we did not observe differences in corticosterone concentration among the groups, however this hormone was measure after colitis induction, that is a chronic stress procedure. It has been shown that chronic stress does not modify either catecholamine or corticoids plasma levels, indicating an adaptation of the neuroendocrine circuit31,32. This strongly suggests that the animals had adapted at the time of the experiment. In conclusion, we here present data that high fat diet (20%) with soybean, fish or a soybean/fish oils mixture did not exacerbate experimental ulcerative colitis in relation to the control diet group (5%). Furthermore, the soybean/fish oils mixture could exert beneficial effects on cytokines balance (IL-10/IL-4 ratio) and protected against DNA damage. The data suggests that this mixture, more than the only fish oil, could contribute as a complementary therapy in colorectal cancer prevention.

Acknowledgments The authors are indebted to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Abbreviation used: AIN, American Institute of Nutrition; C, Control normal fat group; DAI, disease activity index; DHA, docosahexaenoic acid; DSS, dextran sulfate sodium; EPA,

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eicosapentaenoic acid; HF, fish high fat group; HS, Soybean high fat group; HSF, soybean-fish high fat group; IBD, inflammatory bowel disease; IFN-γ, interferongama; IL-10, interleukin-10; IL-4, interleukin-4; IS, inflammation score; MPO, myeloperoxidase; MUFA, monounsaturated fatty acid; Nd, not detectated; Ni, not identificated; NK, natural killer; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; Th1, T helper 1; Th2, T helper 2; TM, Tail moment; UC, ulcerative colitis,

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Tables

Table 1 - Composition of the experimental diets according AIN-9319 Diet( g/100 g) Ingredient

HS

HF

HSF

Casein+

20.0 (14.0)

20.0 (14.0)

20.00 (14.0)

Corn starch*

62.0 (71.1)

62.0 (71.1)

62.00 (71.1)

Cellulose*

5.0 (5.0)

5.0 (5.0)

5.0 (5.0)

Mineral mix AIN-93@

3.5 (3.5)

3.5 (3.5)

3.5 (3.5)

Vitamin mix AIN 93@

1.0 (1.0)

1.0 (1.0)

1.0 (1.0)

Choline bitartrate*

0.25 (0.25)

0.25 (0.25)

0.25 (0.25)

L-cystina*

0.3 (0.18)

0.3 (0.18)

0.3 (0.18)

0.014 (0.007)

0.014 (0.007)

0.014 (0.007)

0

20.0 (20.0)

10.0 (10.0)

20.0 (20.0)

0

10.0 (10.0)

Butylhydroquinone* Fish oil# Soybean oil&

(C)-Control normal fat group, (HS)-Soybean high fat group, (HF)-Fish high fat group and (HSF)-Soybean-fish high fat group. The first number refers to the growth diet (AIN-93 G) and the number in parentheses refers to maintenance diet (AIN-93 M) when its composition differed from that of the growth diet. +casein was obtained from Sinth. *L-cystine, cornstarch, butylhydroquinone, cellulose and choline bitartrate were obtained from Viafarma, São Paulo, Brazil. Oil was supplied from soybean& (Lisa/ind. Brazil) and fish oil# from Campestre Ind. and Com., Brazil. Mineral mix@ and vitamin mix@ AIN-93 from Rhoster Ind. and Com. LTDA.

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Table 2 - Fatty acids composition of the experimental diets Percentage of total fatty acids Fatty acids (%)

C

HS

HF

HSF

16:0

10.2

9.4

20.9

17.9

18:0

6.2

6.8

17.1

5.6

18:1 (w-9)

21.5

25.5

18.4

25.8

18:2 (w-6)

47.4

49.5

7.9

30.1

18:3 (w-3)

3.7

4.2

0.8

2.5

20:4 (w-6)

Nd

Nd

Nd

Nd

20:5 (w-3)

Nd

Nd

16.2

8.2

22:6 (w-3)

Nd

Nd

14.2

6.4

Ni

11

7.0

4.5

4.0

Total SFA

16.4

16.2

38.0

23.5

Total MUFA

21.5

25.5

18.4

25.8

Total PUFA

51.1

49.5

39.1

46.7

W-6 PUFA

47.4

46.8

7.9

32.1

W-3 PUFA

3.7

4.2

31.2

17.1

12:1

11:1

W-6:W-3 PUFA

2:1

(C)-Control normal fat group, (HS)-Soybean high fat group, (HF)-Fish high fat group and (HSF)-Soybean-fish high fat group. SFA; MUFA, monounsaturated fatty acids; PUFA; Nd, not detectated; Ni, not identificated.

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Table 3 - Liver fatty acids incorporation in experimental groups Fatty acids (%)

C

HS

HF

HSF

14:0 16:0

0.8 3.1

0.7 1.8

1.1 19.90

0.8 12.2

18:0

3.6

5.7

7.9

4.6

20:0

0.9

Nd

Nd

0.5

16:1 (w-7)

3.0

2.9

8.0

3.5

18:1 (w-9)

14.6

13.6

12.4

15.7

18:2 (w-6)

15.6

27.0

9.2

23.2

18:3 (w-3)

4.0

7.6

4.1

3.7

20:4 (w-6) 20:5 (w-3)

8.36

12.2

3.4

9.1

4.40

1.77

11.3

6.1

22:6 (w-3)

1.8

1.2

15.3

6.1

Total SFA

8.4

8.2

28.9

18.1

Total MUFA

17.6

16.5

20.4

19.2

Total PUFA Total PUFA w-3

34.16

49.7

43.3

48.2

10.2

10.57

30.7

15.9

Total PUFA w-6

23.96

39.2

12.6

32.3

(C)-Control normal fat group, (HS)-Soybean high fat group, (HF)-Fish high fat group and (HSF)-Soybean-fish high fat group. SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acid; Ns, not detectated;

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Table 4 - Colon length (cm), inflammation score (IS), tissue MPO activity (U/mg) and corticosterone plasma concentration (µg/100mL) in experimental groups

Colon length

Inflammation Score

MPO

14,26 ± 0,24

2,33 ± 0,33

0,84 ± 0,23

HS

14,66 ± 0,45

2,16 ± 0,30

0,67 ± 0,25

HF

15,06 ± 0,14

2,00 ± 0,25

1,17 ± 0,37

14,59 ± 0,66

HSF

15,36 ± 0,12

1,66 ± 0,21

0,95 ± 0,16

14,57 ± 1,48

C

Corticosterone 14,57 ± 1,40

12,23 ± 1,09

(C)-Control normal fat group, (HS)-Soybean high fat group, (HF)-Fish high fat group and (HSF)-Soybean-fish high fat group. Values represent means ± SEM, n= 6.

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Figure 1. Colonic tissue concentration (ρg/mL) of IL-4 (A), IL-10 (B) and INF-y (C) in DSS-induced colitis rats fed with C, HS, HF or HSF diet. Values represent means ± SEM, n=6; * Different (p < 0.05) from the HS group.

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Figure 2. IL-10/IL-4 ratio (anti-inflammatory/pro-inflammatory) of colonic tissue concentration (A) and DNA damage levels (tail moment/100 cells isolated from colon) (B) in DSS-induced colitis rats fed with C, HS, HF or HSF diet. Values represent means ± SEM, n=6; * Different (p < 0.05) from the HS group.

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