Efeitos da exposição subcrônica de Residual Oil Fly Ash (ROFA

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UNIVERSIDADE FEDERAL DE CIÊNCIAS DA SAÚDE DE PORTO ALEGRE – UFCSPA CURSO DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

Marlise Di Domenico

Efeitos da exposição subcrônica de Residual Oil Fly Ash (ROFA) associada à administração de resveratrol

Porto Alegre 2015

Marlise Di Domenico

Efeitos da exposição subcrônica de Residual Oil Fly Ash (ROFA) associada à administração de resveratrol Dissertação submetida ao Programa de Pós-Graduação em Ciências da Saúde da Fundação Universidade Federal de Ciências da Saúde de Porto Alegre como requisito para a obtenção do grau de Mestre

Orientadora: Cláudia Ramos Rhoden

Porto Alegre 2015

DEDICATÓRIA

Às pessoas que me incentivam: Pai, Mãe, Márcia e Mariane

RESUMO

Residual oil fly ash (ROFA) é um poluente do ar comum em áreas em que há queima de óleo. Assim como o material particulado (MP), o ROFA apresenta partículas de diferentes diâmetros que podem ser inaladas pelos seres humanos e causar dano principalmente ao sistema respiratório. O resveratrol (RSV), um polifenol natural, tem recebido cada vez mais atenção devido sua ampla bioatividade, incluindo inibição de tumorgênese, modificação de lipídios e restrição-calórica. O objetivo deste estudo foi investigar a exposição subcrônica ao ROFA e os efeitos da ingestão de RSV em pulmão de ratos. Para isso, trinta e três ratos Wistar machos foram distribuídos nos seguintes grupos: controle (n = 9, CTL), resveratrol (n = 8, RSV), residual oil fly ash (n = 8, ROFA) e ROFA tratados com RVS (n = 8, ROFA + RSV). Os ratos foram expostos ao ROFA por instilação intranasal e foram tratados com RVS (20mg/kg/dia) por gavagem durante 14 semanas. Após vinte e quatro horas, os pulmões foram coletados para a determinação de marcador de dano oxidativo (substâncias reativas ao ácido tiobarbitúrico – TBARS), estado antioxidante (atividade da superóxido dismutase – SOD e da catalase – CAT), dano ao DNA, concentração de metais e de interleucinas. Os resultados demonstram que não houve diferença estatisticamente significativa no TBARS e na atividade de SOD e CAT, na concentração de metais e de interleucinas entre os grupos. O grupo ROFA apresentou maior dano ao DNA quando comparado aos demais grupos. Em conclusão, o RSV exerceu efeito protetor evitando o dano ao DNA após a exposição subcrônica ao ROFA.

Palavras-chave: poluição do ar, antioxidantes, dano ao DNA, estresse oxidativo, inflamação, material particulado.

ABSTRACT

Residual oil fly ash (ROFA) is a common pollutant in areas where there is oil burning. As the particulate matter (PM), the ROFA contains particles from various diameters that can be inhaled by humans and cause damage mainly to the respiratory system. Resveratrol (RSV), a natural polyphenol, has received increasing attention due its varied bioactivities, including the inhibition of tumorigenesis, lipid modification and calorie-restriction. The aim of this study was to investigate the subchronic exposure to ROFA and the effects of RSV intake on rat lungs. For this, thirty-three male Wistar rats were distributed into the following groups: control (n = 9, CTL), resveratrol (n = 8, RSV), residual oil fly ash (n = 8, ROFA) and ROFA plus treatment with RVS (n = 8, ROFA + RSV). The rats were exposed to ROFA by intranasal instillation and were treated with RVS (20mg/kg/day) by gavage for 14 weeks. After twenty four hours, lung tissues were collected for determination of oxidative damage marker (thiobarbituric acid reactive substances - TBARS), antioxidant status (superoxide dismutase – SOD and catalase - CAT activity), DNA damage, cytokines and metals levels. The results show no statistically significant difference in TBARS, SOD and CAT activity, cytokines and metals levels among groups. The ROFA group showed higher DNA damage when compared to the other groups. In conclusion, the present study demonstrated that RSV avoided DNA damage after subchronic ROFA exposure.

Keywords: resveratrol, air pollution, DNA damage, oxidative stress, inflammation, ROFA.

Sumário

Resumo Abstract Lista de abreviaturas 1 INTRODUÇÃO ..................................................................................................... 9 1.1

Poluição Atmosférica ..................................................................................... 9

1.1.1

Definição e dados epidemiológicos .......................................................... 10

1.1.2

Mecanismos fisiopatológicos .................................................................... 12

1.1.3

Residual Oil Fly Ash (ROFA) .................................................................... 15

1.2

Resveratrol .................................................................................................. 17

1.2.1

Origem e propriedades físico-químicas .................................................... 17

1.2.2

Mecanismo de ação ................................................................................. 19

1.2.3

Potencial terapêutico ................................................................................ 21

2 JUSTIFICATIVA ................................................................................................. 23 3 OBJETIVOS ....................................................................................................... 24 3.1 Objetivo Geral ................................................................................................. 24 3.2 Objetivos Específicos ...................................................................................... 24 4 REFERÊNCIAS .................................................................................................. 25 5 ARTIGO............................................................................................................. 36 6 CONSIDERAÇÕES FINAIS ............................................................................... 57 ANEXO A – PARECER DA COMISSÃO DE ÉTICA NO USO DE ANIMAIS (CEUA) .............................................................................................................................. 58 ANEXO B – NORMAS DE PUBLICAÇÃO DA REVISTA....................................... 59

Lista de Abreviaturas

AMPK - 5’ adenosina monofosfato quinase ativada CAT- catalase DEP – partículas de exaustão do diesel DNA – desoxirribonucleic acid / ácido desoxirribonucleico ERN - espécies reativas de nitrogênio ERO - espécies reativas de oxigênio ESCAPE - European Study of Cohorts for Air Pollution Effects / Estudo Europeu de Coorte dos Efeitos da Poluição Atmosférica H2O2 – peróxido de hidrogênio HPAs – hidrocarbonetos policíclicos aromáticos IL-6 – interleucina 6 IL-8 – interleucina 8 IARC – International Agency for Research on Cancer / Agência Internacional de Pesquisa em Câncer MP – material particulado MP10 – material particulado com diâmetro inferior a 10 µm MP2,5 – material particulado com diâmetro inferior a 2,5 µm NAD+ - nicotinamida adenina nucleotídeo NF-κB – factor nuclear kappa B / fator nuclear kappa B NO – óxido nítrico O•-2 - radical superóxido •OH - radical hidroxil ONOO- - peroxinitrito

PUF – partícula ultrafina ROFA - residual oil fly ash RSV - resveratrol SIRT 1 – sirtuina 1 SOD - superóxido dismutase TNF-α – fator de necrose tumoral

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1 INTRODUÇÃO 1.1

Poluição Atmosférica

Poluição atmosférica é um problema de saúde pública que abrange todo o mundo. Estudos epidemiológicos têm demonstrado associação entre a exposição à poluição atmosférica e o aumento de morbidade e mortalidade devido às doenças cardiovasculares e pulmonares, incluindo câncer de pulmão, doença pulmonar obstrutiva crônica e infarto do miocárdio, principalmente em indivíduos suscetíveis (1-5). Os efeitos da poluição sobre a saúde continuam sendo objeto de interesse da comunidade científica e regulatória, além da própria sociedade. Recentemente, a Agência Internacional de Pesquisa em Câncer (IARC - International Agency for Research on Cancer) classificou a poluição atmosférica e o material particulado (MP), proveniente da poluição, como cancerígenos para seres humanos (grupo 1) (6). Apesar de as últimas décadas serem marcadas por avanços tecnológicos importantes que contribuíram para a redução na concentração de emissões de poluentes, este avanço não ocorreu de forma uniforme. Infelizmente, os países em desenvolvimento ainda são os que emitem as maiores concentrações de poluentes à atmosfera (7). Há diferentes fontes de poluição que variam de região para região e, consequentemente, emitem diferentes poluentes. Nos centros urbanos a que prevalece é aquela advinda principalmente dos veículos automotores: da queima de combustíveis fósseis e da biomassa. Assim, estudos têm sido desenvolvidos com o intuito de investigar os mecanismos de ação tóxica dos diferentes compostos presentes na poluição como diesel, biodiesel, MP, hidrocarbonetos policíclicos aromáticos (HPAs), entre outros e seus potenciais efeitos adversos à saúde do ser humano. Diante da complexidade de seus constituintes e mecanismos de ação tóxica que a poluição atmosférica engloba, ainda existem gaps na literatura que precisam

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ser elucidados referentes à maneira pela qual a poluição atmosférica contribui para os problemas de saúde ambiental e humana.

1.1.1 Definição e dados epidemiológicos

A poluição atmosférica é definida como sendo uma mistura física e quimicamente diversificada de partículas e gases provenientes de várias fontes como a queima de combustíveis fósseis, fontes naturais e também pela transformação atmosférica secundária (8). Ela é constituída por materiais carbonáceos (orgânico e elementar), sulfatos, nitratos, carbonatos, metais, peróxidos, quartzo, silicatos (argilas e amianto), óxidos minerais, bactérias, vírus, pólen, detritos animal e vegetal, e MP (9). Além disso, pode apresentar uma variedade de substâncias orgânicas como HPAs, e nitro-HPAs, aldeídos, cetonas, nitro compostos, e quinonas (10). Estudos epidemiológicos demonstram prejuízos à saúde causados pela exposição à poluição do ar em diversos países. Pesquisadores de Harvard observaram, com base em um estudo em seis cidades dos Estados Unidos, que a mortalidade para todas as causas aumenta 14% a cada aumento de 10 µg/m3 na concentração de MP2.5. Além disso, os autores evidenciaram um aumento de 26% em mortes decorrentes de problemas cardiovasculares e de 37% devido a câncer pulmonar para o mesmo aumento de MP2.5. Contrariamente, a diminuição de 2,5 µg/m3 de MP2.5 com duração mínima de um ano foi associada a uma diminuição de 3,5% na mortalidade (11). Na Europa, o projeto Estudo Europeu de Coorte dos Efeitos da Poluição Atmosférica (ESCAPE - European Study of Cohorts for Air Pollution Effects) tem correlacionado a exposição a longo prazo à poluição e efeitos adversos à saúde. De acordo com Raaschou-Nielsen et al. (2013), a exposição ao MP está associada ao risco de câncer de pulmão (4). Além disso, o mesmo grupo de pesquisadores relatou que a exposição de longo prazo às partículas finas está associada a causas naturais de mortes mesmo quando os valores de poluição se encontram abaixo do limite ambiental permitido na Europa (12).

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No Brasil, os níveis ambientais elevados de poluição atmosférica têm sido associados ao aumento de visitas a emergências por causas respiratórias, a internações, e até morte em idosos e crianças (13-16). Dentre os constituintes da poluição atmosférica, o MP tem sido largamente investigado e é caracterizado como uma complexa mistura de componentes orgânicos e inorgânicos dentre os quais destacam-se hidrocarbonetos, metais, sais e ainda microorganismos (17). De acordo principalmente com a fonte emissora e as condições climáticas, o MP pode variar de composição, tamanho e concentração (18). As partículas são classificadas de acordo com seu tamanho aerodinâmico como grossas (2,5–10 μm; PM10), finas (0,1–2,5 μm; PM2.5), e ultrafinas (<100 nm; PUF) (19, 20). O tamanho da partícula é muito importante em termos de saúde, porque se relaciona aos efeitos biológicos no organismo – partículas com maior tamanho aerodinâmico depositam-se quase que exclusivamente no trato respiratório superior enquanto que partículas finas e ultrafinas podem atingir os alvéolos e, consequentemente, adentrar na circulação sistêmica (21, 22). As PUF são potencialmente mais perigosas devido ao seu pequeno tamanho e grande área de superfície, a qual determina o número de grupos reativos na superfície da partícula (20). A poluição atmosférica tem sido correlacionada aos efeitos adversos à saúde. Por sua vez, esses efeitos não se restringem ao sistema respiratório, apesar deste ser o primeiro a entrar em contato. Estudos experimentais relatam que a exposição às partículas presentes na atmosfera pode causar danos sistêmicos: cardiovasculares (2,3), no sistema nervoso (23, 24) e reprodutivo (25). Com

ênfase

ao

sistema

respiratório,

estudos

epidemiológicos

demonstraram uma forte associação entre MP e desenvolvimento de doenças pulmonares como asma e doença pulmonar obstrutiva crônica (26). Além disso, o aumento do risco de câncer de pulmão foi observado até mesmo em áreas onde a concentração de MP2.5 está abaixo da recomendada pelos padrões internacionais mais rígidos (12).

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1.1.2 Mecanismos fisiopatológicos

Nas últimas décadas, os mecanismos fisiopatológicos pelos quais a poluição causa efeitos adversos à saúde humana começaram a ser elucidados. Tanto estudos in vitro quanto in vivo têm relatado efeitos deletérios causados pelos diferentes compostos da poluição, e entre os mecanismos envolvidos na injúria celular destacam-se o estresse oxidativo, a inflamação e o dano ao ácido desexirribonucleico (DNA). As partículas finas, como mencionado, possuem a capacidade de penetrar nas vias respiratórias e de se depositar principalmente nos bronquíolos e alvéolos. Devido à diversidade de componentes presentes nessas partículas como metais de transição, HPAs e compostos orgânicos voláteis entre outros, há a formação de espécies reativas de oxigênio (ERO) e de nitrogênio (ERN). Entre as ERO e ERN destacam-se o peróxido de hidrogênio (H2O2), o radical superóxido (O2.-), o oxigênio singlet (1O2), o radical hidroxil (̇.OH), óxido nítrico (NO), peroxinitrito (ONOO-), os quais são capazes de interagir com lipídios, proteínas e DNA e, consequentemente, causar dano e morte celular (27-30). Os estudos de exposição aguda aos diferentes constituintes de poluição atmosférica têm relacionado principalmente o estresse oxidativo e a inflamação das vias aéreas como uma primeira resposta à exposição. O estresse oxidativo é caracterizado por um desequilíbrio entre substâncias antioxidantes e próoxidantes, onde há um deslocamento para o estado pró-oxidante. De maneira geral, as ERO e ERN são produtos naturais do metabolismo e são importantes para determinados eventos celulares como, por exemplo, transdução de sinal, ativação enzimática, expressão gênica, entre outros (31, 32). As células possuem um sistema enzimático de defesa antioxidante endógeno capaz de protegê-las contras as ERO, sendo que as principais são a superóxido dismutase (SOD), a catalase, (CAT) e o sistema redox da glutationa (glutationa peroxidase e glutationa S-tranferase). Em condições fisiológicas, essas enzimas são importantes na

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proteção do dano oxidativo, como lipoperoxidação, oxidação de proteínas e dano ao DNA, pois são capazes de interceptar, neutralizar e reagir com intermediários gerados em excesso pelas ERO e ERN (33-35). Entretanto, pode haver uma exacerbação de ERO devido a um estímulo não fisiológico, como a exposição a poluentes, culminando no estresse oxidativo. O processo de inflamação, após a exposição a xenobióticos, resulta em uma complexa sequência de eventos que objetivam remover a fonte de inflamação e resolver a reação inflamatória. Esse processo é regulado por mediadores que são secretados pelos tecidos e células inflamatórias e variam de acordo com o tipo e as propriedades da partícula, como solubilidade e superfície reativa (36). Estudos experimentais revelaram que após a exposição aos diferentes constituintes da poluição atmosférica há aumento dos biomarcadores de inflamação, tais como a ativação do fator nuclear kappa B (NF-κB), liberação de citocinas, além do aumento da produção de óxido nítrico (NO). Em um trabalho de revisão, Moller et al. (2015), observaram que há diferenças na resposta inflamatória causada pela exposição dependendo do local do estudo (37). Os autores explicam que isso está associado à variação – de tempo, espaço e condições climáticas - dos constituintes presentes na atmosfera no período de realização dos estudos além da diversidade dos modelos de exposição utilizados que podem variar quanto ao tempo de exposição e tipo de poluente. Os resultados de estudos in vitro e in vivo são controversos quando relacionam o tamanho da partícula ao potencial efeito inflamatório. A relação de que quanto maior a partícula maior será a resposta inflamatória é evidenciada em alguns estudos, contrária em outros, e outros ainda afirmam que há uma resposta inflamatória similar entre os diversos tamanhos de partículas (38-41). Além do tamanho e composição, deve-se ter em consideração a solubilidade dos compostos presentes na partícula, pois pode ser um fator importante em relação aos efeitos inflamatórios. A fração solúvel em água de MP10, de partículas ambientais e residual oil fly ash (ROFA) gera níveis mais elevados de secreção de citocinas do que a fração insolúvel (42).

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As diferenças de resposta inflamatória podem ser explicadas pelo estresse oxidativo gerado pelos metais de transição presentes nas partículas. Estudos que utilizaram tratamentos com quelantes de metais de transição apresentaram diminuição da secreção de citocinas – interleucina 6 (IL-6), interleucina 8, (IL-8) e fator de necrose tumoral (TNF-α) (43, 44). A instilação aguda de partículas provenientes da queima do diesel causa inflamação pulmonar, que pode ser caracterizada pelo influxo de células inflamatórias, aumento de proteínas totais, e estresse oxidativo (45-47). Já partículas de

MP10 podem induzir

uma

resposta inflamatória

sistêmica

caracterizada pelo aumento de mediadores pró-inflamatórios sistêmicos como IL-6 (48). Além do estresse oxidativo e da inflamação, se tornou evidente a relação entre exposição à poluição atmosférica e danos ao DNA. O potencial carcinogênico das partículas pode estar vinculado a duas principais vias genotóxicas: a via primária e a via secundária (36, 49, 50). A via secundária pode ser proveniente de um dano genético resultante de excessiva e persistente formação de ERO/ERN geradas durante o processo de inflamação provocado por fagócitos ativados ou pela reação de Fenton devido à presença de metais (49, 51). Nessa via, as ERO/ERN podem causar dano ao DNA por mutações, deleções, ou inserções. A via primária, por sua vez, é caracterizada como um dano genético causado pelas partículas na ausência de inflamação. Esse dano ocorre por mecanismos diretos e indiretos que também variam de acordo com as propriedades físicas e químicas das partículas. De forma direta, os componentes presentes nas partículas podem interagir com o DNA genômico, e de forma indireta os metais e/ou constituintes orgânicos presentes podem formar adutos de DNA (36). A literatura é vasta a respeito da associação entre exposição à poluição do ar e dano ao DNA. Tanto estudos in vitro quanto in vivo demonstram aumento de 8-dihidro-2-deoxiguanosina (8-oxodG), formado pela oxidação da guanina ou incorporação durante a replicação ou reparo como nucleotídeo oxidado, após a

15

exposição a MP, partículas de exaustão do diesel (DEP), ROFA (52-56). Estudos em humanos também demonstram essa associação (57). Entretanto, é importante ressaltar que a célula possui mecanismos de reparo que podem reverter o dano causado pela exposição a compostos mutagênicos como reparo por excisão de base (BER), reparo por excisão de nucleotídeo (NER), entre outros (58). Além desses mecanismos, o organismo tem a capacidade de se manter em homeostase sendo capaz de, quando necessário, eliminar uma célula não desejada ou danificada através do processo de apoptose (59). Portanto, devido à elevada complexidade e variação dos componentes da poluição não é uma tarefa fácil se distinguir qual dos componentes provocaria o dano ao DNA. Diante disso e da complexidade envolvida na carcinogênese (o dano ao DNA representa apenas um de todos os complexos processos envolvidos na carcinogênese) é importante que a interpretação e extrapolação dos resultados sejam realizados cuidadosamente. 1.1.3 Residual Oil Fly Ash (ROFA)

O ROFA tem sido utilizado em estudos como um substituto para partículas de poluição atmosférica, uma vez que ele é rico em metais e sua constituição pode ser precisamente determinada. ROFA é o termo utilizado para se referir aos resíduos principalmente inorgânicos que permanecem após a oxidação incompleta de compostos de carbono (60). Geralmente as partículas de ROFA possuem tamanho menor do que 2,5 µm e são quimicamente consideradas complexas quando comparadas às demais partículas de poluição atmosférica; pode apresentar sulfatos, silicatos, carbono e nitrogênio contendo outros componentes, contaminantes fósseis e outros aditivos. Além dos elementos citados, também apresenta uma grande quantidade de metais que estão naturalmente presentes em combustíveis (petróleo, parafina, e óleo diesel) e permanecem quando a fração volátil é destilada.

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Apesar de o ROFA não mimetizar a poluição atmosférica como um todo, este MP contém elevadas concentrações de poluentes que são encontrados na poluição do ar. Devido ao fato de o ROFA ser rico em metais, muitos trabalhos têm utilizado ele como um substituto de MP ambiental para avaliar os efeitos biológicos mediados pelos metais presentes nos poluentes atmosféricos. O ROFA possui frações solúveis e não solúveis em água. Estudos relatam que os efeitos causados por ambas as frações são similares, sendo que os componentes solúveis causam um dano mínimo no pulmão em ratos expostos ao composto (61). Já Roberts et al. (2003), observou que a fração solúvel do ROFA aumenta a susceptibilidade à infecção pulmonar em ratos que receberam uma dose aguda das diferentes frações de ROFA (62). Essas variações na resposta podem ser explicadas pelas diferenças de composição, concentração e tempo de exposição. A exposição aguda a este poluente tem demonstrado que ele é capaz de causar dano pulmonar em modelos experimentais. Muitos trabalhos têm associado o dano pulmonar à presença de metais na constituição do ROFA (44, 63, 64). Os primeiros estudos revelam que os metais inalados catalisam reações químicas de Fenton e, consequentemente, há produção de ERO (60). As ERO são capazes de induzir a expressão de citocinas pró-inflamatórias IL-6, IL-8 e TNFα em cultura de célula do epitélio respiratório humano, e também de antioxidantes (44). Recentemente, alguns trabalhos têm sugerido uma relação tempo dependente capaz de explicar as diferentes respostas celulares após a instilação aguda ao ROFA. Magnani et al. (2011), observaram que no pulmão, inicialmente, ocorre oxidação fosfolipídica e o dano às proteínas se dá após transcorrer um período maior de tempo após a exposição (65). O mesmo grupo de pesquisadores verificou resposta semelhante em outros órgãos, o que demonstra efeitos sistêmicos à instilação de ROFA (66-68). Além disso, também foi observada uma resposta semelhante em relação às citocinas pró-inflamatórias. No pulmão, o TNFα e a IL-6 estão presentes em uma maior concentração 3 horas após a exposição e em menor concentração após cinco horas (67). De maneira semelhante, Carvalho et al. (2014), observaram um aumento de colapso alveolar, influxo de

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células polimorfonucleares, alterações ultraestruturais no parênquima e aumento de neutrófilos sanguíneos nas primeiras 6, 24, 48, 72 e 96 horas, sendo que estes desfechos retornaram aos valores normais após 120 horas da exposição ao ROFA (69). Corroborando com os dados, pesquisadores verificaram que a exposição aguda a este poluente causa lipoperoxidação no pulmão, mas esse perfil não foi observado na exposição crônica (70, 71). Esses dados sugerem adaptação tecidual ao estresse. Com o intuito de avaliar os efeitos protetores na resposta celular em organismos expostos ao ROFA algumas substâncias antioxidantes têm sido utilizadas. Rhoden et al. (2004), demonstrou que a utilização de N-acetilcisteína, um antioxidante, impediu o desenvolvimento de processo inflamatório e de estresse oxidativo em modelo experimental de poluição do ar (72). De maneira semelhante, a administração sistêmica de dimetiluréia, um scavenger de uma variedade de espécies de oxigênio, demonstrou redução na citotoxicidade e na inflamação pulmonar além de aumento na atividade antioxidante causada pela exposição ao ROFA (62, 73). Isso sugere que o estresse oxidativo desempenha um papel importante no mecanismo de dano pulmonar produzido por este tipo de partícula. Diante disso, os mecanismos responsáveis por desencadear dano pulmonar não são totalmente compreendidos e podem variar de acordo com as propriedades físico-químicas do ROFA, do tempo de exposição e de outros fatores.

Além

disso,

substâncias

antioxidantes

podem

interferir

e,

consequentemente, minimizar e/ou prevenir possíveis danos celulares.

1.2

Resveratrol

1.2.1 Origem e propriedades físico-químicas

Na década de 90, o intitulado “Paradoxo Francês” chamou atenção para a até então, pouco conhecida, substância resveratrol (RSV). Esse paradoxo se

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estabeleceu a partir de estudo epidemiológico, o qual evidenciou que os franceses apresentavam baixa incidência de doenças coronárias apesar de possuírem uma dieta com elevado índice de gordura saturada (74). Isso foi associado ao consumo diário de vinho população. Em 2009, a Organização Mundial da Saúde divulgou dados que corroboram essa evidência: a proporção de morte relacionada à doença coronária na França foi de duas a três vezes menor do que a de outros países, tais como Estados Unidos, Reino Unido e Suécia (75). A partir disso, milhares de estudos começaram a ser desenvolvidos com o objetivo de avaliar os diferentes efeitos desta substância. O RSV apresenta-se naturalmente em duas formas isoméricas: cis e transresveratrol (Figura 1), sendo que o trans-resveratrol (3,5’4-trihidroxiestilbeno) é a forma mais estável. É produzido por uma grande variedade de plantas incluindo uvas, amendoim, berries, e nozes como um mecanismo de defesa em reposta ao estresse, a injúria ou ao ataque de microrganismos (76, 77). Como outros polifenóis, ele tem sido associado a vários processos biológicos como, por exemplo, sendo um potente antioxidante, cardioprotetor, anticancerígeno e capaz de modular a atividade anti-inflamatória (78).

A

B

Figura 1. Estrutura química dos isômeros trans-resveratrol (A) e cisresveratrol (B).

A maioria desses efeitos tem sido demonstrada em estudos realizados in vitro. Estudos realizados em modelos in vivo, tanto em animais como em humanos, demonstram que esses efeitos são difíceis de serem reproduzidos. Uma

19

plausível explicação para essa discrepância se deve, principalmente, a baixa biodisponibilidade do RSV quando administrado oralmente in vivo (79, 80). Após a administração, o RSV é rapidamente metabolizado em conjugados glicurônicos e sulfatos. Em humanos, o pico de concentração ocorre após 30 minutos da ingestão, enquanto que em animais o tempo de meia vida se dá entre 12-15 minutos após a administração oral (81, 82). Entretanto, devido ao perfil lipofílico, a concentração nos tecidos deve ser maior do que a do plasma (77). Muitos dos efeitos relatados são devido à ação dos metabólitos do RSV já que a concentração e tempo de meia vida são maiores nos metabólitos do que a própria substância (83).

1.2.2 Mecanismo de ação

Muitos estudos têm sido desenvolvidos a fim de elucidar o mecanismo de ação do RSV. De maneira geral, a literatura demonstra que essa substância regula a função biológica por meio de proteínas-alvo específicas: as sirtuinas, particularmente a sirtiuna 1 (SIRT1), e a 5’ adenosina monofosfato quinase ativada (AMPK). As sirtuinas, pertencentes a uma família de genes altamente conservada, removem grupos de acetil das proteínas e os transferem para a nicotinamida adenina nucleotídeo (NAD+), e são reconhecidas por estarem envolvidas em múltiplos processos biológicos associados ao metabolismo energético e a respostas ao estresse como, por exemplo, isquemia (84). Existem relatos controversos referentes à ativação da SIRT1 pelo RSV: enquanto que alguns estudos demonstram que as sirtuinas são ativadas pelo RSV (85) outros não relatam essa dependência (86). Já o AMPK pode ser um alvo direto do RSV e sua atividade é modulada pelo aumento da razão de AMP/ATP, a qual reflete o estado de energia da célula. (87, 88). Geralmente, o AMPK é ativado em condições fisiológicas de estresse energético como hipóxia, exercício e jejum (89, 90). Estudos demonstram que o

20

AMPK é essencial para os efeitos metabólicos do RSV e é ativado independentemente de SIRT1 (91). Entretanto, também há estudos que demonstram diminuição da atividade de AMPK quando há inibição da atividade de SIRT1 (92). Além das sirtuinas e do AMPK, sabe-se que o RSV tem a capacidade de interagir com um grande número de receptores, quinases e outras enzimas, os quais contribuem para distintos efeitos biológicos. Estudos têm sugerido que o RSV pode induzir a expressão de enzimas antioxidantes (77), promover a produção de óxido nítrico, inibir a agregação plaquetária, aumentar o colesterol HDL (93, 94), além de diminuir a inflamação e ser capaz de prolongar a vida de mamíferos metabolicamente comprometidos (88). Ainda, estudos demonstram que ele interfere na resistência à insulina e diabetes e é capaz de prevenir a obesidade (95, 96). Além dos efeitos apresentados, a literatura tem demonstrado que no câncer o RSV age através de múltiplos mecanismos, entre eles, proapoptóticos, antiproliferativos,

anti-inflamatórios,

antiangiogênicos

(97).

Os

efeitos

antiproliferativos e inibitórios tem sido atribuídos à habilidade de bloquear a síntese de DNA e interferir em vários estágios do ciclo celular. Essa substância é capaz de estimular o reparo no DNA aumentando a atividade da p53 (98) ou estimulando a eficiência de reparo do DNA nas células com dano (99). Além disso, é capaz de induzir apoptose em muitas linhagens celulares de tumor por meio da ativação de vias intrínsecas e extrínsecas dos mecanismos envolvidos na morte celular (100-102). Apesar de o mecanismo terapêutico não estar completamente elucidado, estudos têm demonstrado efeitos benéficos dessa substância em diferentes sistemas. De maneira geral, devido as características apresentadas o RSV é capaz de prevenir uma grande variedade de doenças, incluindo doenças cardiovasculares (103), câncer (104), dano isquêmico (94, 105), entre outras.

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1.2.3 Potencial terapêutico

O RSV é uma substância natural que tem sido extensivamente estudada desde a sua descoberta. Estudos realizados in vivo e in vitro tem demonstrado que ele exerce diferentes efeitos, os quais podem prevenir ou retardar a progressão de muitas condições patológicas. O RSV é neuroprotetor em modelo de injúria cerebral apresentando atividade antiapoptótica (106, 107) antioxidante e anti-inflamatória (107-109). Além disso, apresenta efeitos benéficos em doenças neurodegenerativas e no dano cognitivo (110-113). Além dos efeitos cerebrais, essa substância também tem sido vinculada a cardioproteção. O RSV foi capaz de prevenir a produção de ERO e de estresse oxidativo, além de preservar a atividade das enzimas antioxidantes, aumentar a resistência vascular e apresentar efeitos antiapoptóticos em células cardíacas e em modelos de isquemia e reperfusão (114, 115). Estudos in vitro demonstram que o RSV pode agir como anticancerígeno e antimutagênico. Estudos têm relatado o potencial protetor do RSV contra câncer de mama (63), gástrico (116), coloretal (117) e outros tipos de cânceres, como o de pulmão e hepático (118, 119). A inibição da proliferação celular anormal via modulação da progressão do ciclo celular é uma das mais importantes estratégias para a quimioproteção e quimioterapia (120). No pulmão, estudos têm demostrado que substâncias com características antioxidantes são capazes de modular mecanismos de defesa. A quercitina, um flavonóide polifenólico com propriedade antioxidante, antiapoptótica entre outras, demonstrou ser efetiva no tratamento ao dano pulmonar causado pela administração de ácido, o qual causa lesão pulmonar química aguda. A quercitina foi capaz de diminuir a infiltração de células inflamatórias e a expressão de óxido nítrico sintase e aumentar a atividade da SOD (121). De maneira semelhante, o eugenol, o qual também possui ações anti-inflamatórias e antioxidantes, diminuiu os danos pulmonares advindos da exposição às partículas de diesel sendo capaz de diminuir as células polimorfonucleares além de reduzir a morte celular, mas não o estresse oxidativo (122).

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Por outro lado, estudos investigaram o efeito do RSV sobre o dano causado pela exposição ao cigarro. O cigarro é constituído por mais de 4000 compostos dentre os quais se destacam os aldeídos, a nicotina, o monóxido de carbono, o material particulado, entre outros (123). Dessa forma, o cigarro é considerado um composto

potencialmente

nocivo

à

saúde

que

pode

causar

doenças

principalmente no sistema respiratório. O RSV foi capaz de atenuar o dano oxidativo causado pelo cigarro por meio da diminuição da secreção de IL-6 e TNF-α e de malondealdeído, além de aumento da atividade de CAT, SOD, GSH-Px nos camundongos expostos. Isso pode ser explicado devido a inibição da ativação de NF-kB (124). Corrobar com as evidências, Kurus et al. (2009), verificou que essa substância tratou e preveniu as alterações histopatológicas na traqueia de ratos expostos à fumaça de cigarro (125). Apesar de o RSV apresentar um efeito positivo da função do ventrículo esquerdo, não foi capaz de prevenir os efeitos deletérios no plasma e no lavado broncoalveolar de porcos causados devido à exposição à fumaça do cigarro (126). Estudo conduzido por Zhang et al. (2014), demonstrou que o RSV foi capaz de proteger o tecido pulmonar em camundongos expostos ao lipossacarídeo, substância capaz de causar endotoxemia. O RSV reverteu o estresse oxidativo evidenciado pela diminuição de biomarcadores pró-oxidantes (MDA e H2O2) e aumento de biomarcadores antioxidantes (CAT, SOD, razão GSH/GSSG), além de inibir a produção de NO e expressão de óxido nítrico sintase em camundongos que receberam lipossacarídeo (127). Diante disso, se evidencia que um significante progresso na compreensão dos mecanismos celulares modulados pelo RSV foi alcançado. Contudo, ainda há necessidade de se elucidar as vias específicas de ativação e manutenção das funções biológicas, das defesas antioxidantes celulares e também, dos mecanismos envolvidos no reparo macromolecular.

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2

JUSTIFICATIVA

Nas últimas décadas é crescente a preocupação acerca dos efeitos adversos da poluição atmosférica à saúde humana. A população das grandes cidades não tem como evitar a exposição à poluição atmosférica, uma vez que ela está constantemente presente na atmosfera, e tão pouco existe antídoto capaz de reverter ou minimizar as consequências advindas da exposição à mesma. Em contrapartida, sabe-se que uma alimentação saudável e balanceada é essencial para a manutenção da vida. Uma das maneiras de se reduzir o extresse oxidativo é reduzir a ingestão calórica selecionando-se alimentos apropriados, principalmente porque os nutrientes compreendem um aspecto importante no sistema de defesa antioxidante. O resveratrol, por exemplo, é uma substância que apresenta propriedades antioxidantes, entre outras, e é encontrado em alimentos de fácil acesso como, por exemplo, a uva. Até o presente momento não há estudos que correlacionam os efeitos do resveratrol com os efeitos deletérios consequentes a exposição à poluição atmosférica. Diante disso, verifica-se a importância de se analisar as consequências da exposição à poluição atmosférica e o potencial efeito terapêutico/protetor desse antioxidante.

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3 OBJETIVOS

3.1 Objetivo Geral

Avaliar parâmetros de estresse oxidativo, de inflamação e de dano ao DNA no pulmão de ratos Wistar machos após a exposição subcrônica ao ROFA e os efeitos da associação com o resveratrol.

3.2 Objetivos Específicos



Determinar os danos oxidativos no pulmão através da determinação de Espécies Reativas ao Ácido Tiobarbitúrico (TBA-RS);



Determinar a atividade das enzimas antioxidantes Catalase (CAT) e Superóxido dismutase (SOD) no pulmão;



Determinar concentração de citocinas pro-inflamatórias IL-6, TNF-α no pulmão através da imumohistoquímica;



Determinar a concentração de metias no pulmão através de espectrometria de massa com plasma acoplado indutivamente (ICP-MS);



Avaliar os danos ao DNA no tecido pulmonar através do ensaio cometa.

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69. Carvalho GM, Nagato LK, Fagundes Sda S, Dos Santos FB, Calheiros AS, Malm O, et al. Time course of pulmonary burden in mice exposed to residual oil fly ash. Front Physiol. 2014;5:366. 70. Petry, M. R. Análise dos efeitos pulmonares e cardiovasculares da inalação aguda de residual oil fly ash em ratos submetidos ao treinamento físico. Dissertação. Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre-RS. 2011. 71. Damiani RM, Piva MO, Petry MR, Saldiva PH, Tavares Duarte de Oliveira A, Rhoden CR. Is cardiac tissue more susceptible than lung to oxidative effects induced by chronic nasotropic instillation of residual oil fly ash (ROFA)? Toxicol Mech Methods. 2012;22(7):533-9. 72. Rhoden CR, Lawrence J, Godleski JJ, González-Flecha B. N-acetylcysteine prevents lung inflammation after short-term inhalation exposure to concentrated ambient particles. Toxicol Sci. 2004;79(2):296-303. 73. Dye JA, Adler KB, Richards JH, Dreher KL.Epithelial injury induced by exposure to residual oil fly-ash particles: role of reactive oxygen species? Am J Respir Cell Mol Biol. 1997;17(5):625-33. 74. Catalgol B, Batirel S, Taga Y, Ozer NN. Resveratrol: French paradox revisited. Front Pharmacol. 2012;17(3):141. 75. World Health Organization. Atlas of global epidemic of heart disease and stroke. Part Three: The Burden. Deaths from Coronary Heart Disease. 2009. 76. Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov. 2006;5(6):493‐506. 77. Timmers S, Auwerx J, Schrauwen P. The journey of resveratrol from yeast to human. Aging (Albany NY). 2012;4(3):146-58. 78. Lastra CA, Villegas I. Resveratrol as an anti-inflammatory and anti-aging agent: mechanisms and clinical implications. Mol Nutr Food Res. 2005;49(5):405–30. 79. Walle T. Bioavailability of resveratrol. Ann N Y Acad Sci. 2011;1215:9-15. 80. Delmas D, Aires V, Limagne E, Dutartre P, Mazué F, Ghiringhelli F. Transport, stability, and biological activity of resveratrol. Ann N Y Acad Sci. 2011;1215:48-59. 81. Goldberg DM, Yan J, Soleas GJ. Absorption of three wine-related polyphenols in three different matrices by healthy subjects. Clin Biochem. 2003;36(1);79–87.

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82. Gescher AJ, Steward WP. Relationship between mechanisms, bioavailibility, and preclinical chemopreventive efficacy of resveratrol: a conundrum. Cancer Epidemiol. Biomarkers Prev. 2003;12(10);953–7. 83. Polycarpou E, Meira LB, Carrington S, Tyrrell E, Modjtahedi H, Carew MA. Resveratrol 3-O-d-glucuronide and resveratrol 40-O-d-glucuronide inhibit colon cancer cell growth: evidence for a role of A3 adenosine receptors, cyclin D1 depletion, and G1 cell cycle arrest. Mol Nutr Food Res. 2013;57(10):1708-17. 84. Bordone L, Guarente L. Calorie restriction, SIRT1 and metabolism: understanding longevity. Nat Rev Mol Cell Biol. 2005;6(4):298–305. 85. Mohar DS, Malik S. The Sirtuin System: The Holy Grail of Resveratrol? J Clin Exp Cardiolog. 2012;3(11):216. 86. Pacholec M, Bleasdale JE, Chrunyk B, Cunningham D, Flynn D, Garofalo RS, et al. SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J Biol Chem. 2010;285:8340–51. 87. Dasgupta B, Milbrandt J. Resveratrol stimulates AMP kinase activity in neurons. Proc Natl Acad Sci USA. 2007;104(17):7217–22. 88. Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature. 2006;444(7117):337–42. 89. Long YC, Zierath JR. AMP-activated protein kinase signaling in metabolic regulation. J Clin Invest. 2006;116(7):1776–83. 90. Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol. 2012;13(4):251–62. 91. Um JH, Park SJ, Kang H, Yang S, Foretz M, McBurney MW, et al. AMPactivated protein kinase-deficient mice are resistant to the metabolic effects of resveratrol. Diabetes. 2010;59(3):554–63. 92. Suchankova G, Nelson LE, Gerhart-Hines Z, Kelly M, Gauthier MS, Saha AK, et al. Concurrent regulation of AMP-activated protein kinase and SIRT1 inmammalian cells. Biochem Biophys Res Commun. 2009;378(4):836–41. 93. Fremont L. Biological effects of resveratrol. Life Sci. 2000;66(8):663-73. 94. Sinha K, Chaudhary G, Gupta YK. Protective effect of resveratrol against oxidative stress inmiddle cerebral artery occlusionmodel of stroke in rats. Life Sci. 2002;71(6):655-65.

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95. MC, Lajoie C, Clement R, Gosselin H, Calderone A, Perrault LP. Female rats fed a high-fat diet were associatedwith vascular dysfunction and cardiac fibrosis in the absence of overt obesity and hyperlipidemia: therapeutic potential of resveratrol. J Pharmacol Exp Ther. 2008;325(3):961–8. 96. Kim S, Jin Y, Choi Y, Park T. Resveratrol exerts anti-obesity effects via mechanisms involving down-regulation of adipogenic and inflammatory processes in mice. Biochem Pharmacol. 2011;81(11):1343–51. 97. Singh CK, Ndiaye MA, Ahmad N. Resveratrol and cancer: Challenges for clinical translation. Biochim Biophys Acta. 2014; S0925-4439(14)00334-2. 98. Huang C, Ma WY, Goranson A, Dong Z. Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway. Carcinogenesis. 1999;20(2):237-42. 99. Chakraborty S, Roy M, Bhattacharya RK. Prevention and repair of DNA damage by selected phytochemicals as measured by single cell gel electrophoresis. J Environ Pathol Toxicol Oncol. 2004; 23(3):215-26. 100. Kundu JK, Surh YJ. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives. Cancer Lett. 2008;269(2):243-61. 101. Garvin S, Ollinger K, Dabrosin C. Resveratrol induces apoptosis and inhibits angiogenesis in human breast cancer xenografts in vivo. Cancer Lett. 2006;231(1):113–122. 102. Kalra N, Roy P, Prasad S, Shukla Y. Resveratrol induces apoptosis involving mitochondrial pathways in mouse skin tumorigenesis. Life Sci. 2008;82(7-8):348– 58. 103. Bradamante S, Barenghi L, Villa A. Cardiovascular protective effects of resveratrol. Cardiovasc Drug Rev. 2004;2(3):169-88. 104. Carter LG, D'Orazio JA, Pearson KJ. Resveratrol and cancer: focus on in vivo evidence. Endocr Relat Cancer. 2014;21(3):R209-25. 105. Wang Q, Xu J, Rottinghaus GE, Simonyi A, Lubahn D, Sun GY, Sun AY. Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res. 2002; 958(2):439-47. 106. Zhou XM, Zhou ML, Zhang XS, Zhuang Z, Li T, Shi JX, et al. Resveratrol prevents neuronal apoptosis in an early brain injury model. J Surg Res. 2014;189(1):159-65.

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107. Mohamed HE, El-Swefy SE, Hasan RA, Hasan AA. Neuroprotective effect of resveratrol in diabetic cerebral ischemic-reperfused rats through regulation of inflammatory and apoptotic events. Diabetol Metab Syndr. 2014;6(1):88. 108. Orsu P, Murthy BV, Akula A. Cerebroprotective potential of resveratrol through anti-oxidant and anti-inflammatory mechanisms in rats. J Neural Transm. 2013;120(8):1217-23. 109. Li J, Feng L, Xing Y, Wang Y, Du L, Xu C, et al. Radioprotective and antioxidant effect of resveratrol in hippocampus by activating Sirt1. Int J Mol Sci. 2014;15(4):5928-39. 110. Richard T, Pawlus AD, Iglésias ML, Pedrot E, Waffo-Teguo P, Mérillon JM, et al. Neuroprotective properties of resveratrol and derivatives. Ann N Y Acad Sci. 2011;1215:103-8. 111. Sun AY, Wang Q, Simonyi A, Sun GY. Resveratrol as a therapeutic agent for neurodegenerative diseases. Mol Neurobiol. 2010;41(2-3):375-83. 112. Renaud J, Martinoli MG. Resveratrol as a protective molecule for neuroinflammation: a review of mechanisms. Curr Pharm Biotechnol. 2014;15(4):318-29. 113. Anastácio JR, Netto CA, Castro CC, Sanches EF, Ferreira DC, Noschang C, et al. Resveratrol treatment has neuroprotective effects and prevents cognitive impairment after chronic cerebral hypoperfusion. Neurol Res. 2014;36(7):627-33. 114. Mokni M, Hamlaoui S, Karkouch I, Amri M, Marzouki L, Limam F, et al. Resveratrol provides cardioprotection after ischemia/reperfusion injury via modulation of antioxidant enzyme activities. Iran J Pharm Res. 2013;12(4):867-75. 115. Ungvari Z, Orosz Z, Rivera A, Labinskyy N, Xiangmin Z, Olson S, et al. Resveratrol increases vascular oxidative stress resistance. Am J Physiol Heart Circ Physiol. 2007;292(5):H2417-24. 116. Atten MJ, Godoy-Romero E, Attar BM, Milson T, Zopel M, Holian O. Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells. Invest New Drugs. 2005; 23(2):111–9. 117. Juan ME, Alfaras I, Planas JM. Colorectal cancer chemoprevention by transresveratrol. Pharmacol Res. 2012; 65(6):584–91. 118. Malhotra A, Nair P, Dhawan DK. Curcumin and resveratrol synergistically stimulate p21 and regulate cox-2 by maintaining adequate zinc levels during lung carcinogenesis. Eur J Cancer Prev. 2011;20(5):411–416.

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119. Howells LM, Berry DP, Elliott PJ, Jacobson EW, Hoffmann E, Hegarty B, Brown K, et al. Phase I randomized, double-blind pilot study of micronized resveratrol (SRT501) in patients with hepatic metastases-safety, pharmacokinetics, and pharmacodynamics. Cancer Prev Res. 2011;4(9):1419–25. 120. Kundu JK, Surh YJ. Molecular basis of chemoprevention by resveratrol: NF-jB and AP-1 as potential targets. Mutat Res. 2004;555(1-2):65-80. 121. Yilmaz MZ, Guzel A, Torun AC, Okuyucu A, Salis O, Karli R, et al. The therapeutic effects of anti-oxidant and anti-inflammatory drug quercetin on aspiration-induced lung injury in rats. J Mol Histol. 2014;45(2):195-203. 122. Zin WA, Silva AG, Magalhães CB, Carvalho GM, Riva DR, Lima CC, et al. Eugenol attenuates pulmonary damage induced by diesel exhaust particles. J Appl Physiol. 2012;112(5):911-7. 123. Facchinett F, Amadei F, Geppetti P, Tarantini F, Di Serio C, Dragotto A, et al. Alpha,beta-unsaturated aldehydes in cigarette smoke release inflammatory mediators from human macrophages. Am J Respir Cell Mol Biol. 2007;37(5):617– 23. 124. Liu H, Ren J, Chen H, Huang Y, Li H, Zhang Z, et al. Resveratrol protects against cigarette smoke-induced oxidative damage and pulmonary inflammation. J Biochem Mol Toxicol. 2014;28(10):465-71. 125. Kurus M, Firat Y, Cetin A, Kelles M, Otlu A. The effect of resveratrol in tracheal tissue of rats exposed to cigarette smoke. Inhal Toxicol. 2009;21(12):97984. 126. Al-Dissi AN, Weber LP. Resveratrol preserves cardiac function, but does not prevent endothelial dysfunction or pulmonary inflammation after environmental tobacco smoke exposure. Food Chem Toxicol. 2011;49(7):1584-91. 127. Zhang HX, Duan GL, Wang CN, Zhang YQ, Zhu XY, Liu YJ. Protective effect of resveratrol against endotoxemia-induced lung injury involves the reduction of oxidative/nitrative stress. Pulm Pharmacol Ther. 2014;27(2):150-5.

36

5 ARTIGO

O presente artigo foi elaborado em língua estrangeira e está formatado conforme as normas da revista Toxicology and Applied Pharmacology (Fator de Impacto: 3,630 (2015).

37

Title: Resveratrol prevents pulmonary DNA damage induced by subchronic exposure to residual oil flay ash (ROFA) Marlise Di Domenicoa, Natália de Souza Xavier Costab, Fernando Barbosa Jrc, Gabriel Ribeiro Jrb, Paulo Hilário Nascimento Saldiva b, Cláudia Ramos Rhodena

Affiliation a

Laboratório de Estresse Oxidativo e Poluição Atmosférica, Universidade Federal de

Ciências da Saúde de Porto Alegre, Brazil. b

Laboratório de Poluição Atmosférica Experimental, Faculdade de Medicina da

Universidade de São Paulo, Brazil. c

Laboratório de Toxicologia e Essencialidade de Metais, Universidade de São Paulo,

Ribeirão Preto, Brazil.

Corresponding Author: Cláudia Ramos Rhoden Rua Sarmento Leite, 245, Porto Alegre, RS, Zip-code: 90050-170, Brazil. Tel: 55 51 33038800; Fax: 55 51 33333144; E-mail address: [email protected]

38 1

ABSTRACT

2 3 4

Residual oil fly ash (ROFA) is a common pollutant in areas where there is oil

5

burning. This particulate matter (PM) with wide distribution of particle diameters can be

6

inhaled by human beings and therefore can cause damage to the respiratory system.

7

Resveratrol (RSV), a natural polyphenol, has received increasing attention due its wide

8

bioactivities, including the inhibition of tumorigenesis, lipid modification and calorie-

9

restriction. The aim of this study was to investigate the subchronic exposure to ROFA and

10

the effects of RSV intake on rat lungs. For this, thirty-three male Wistar rats were

11

distributed into the following groups: control (n = 9, CTL), resveratrol (n = 8, RSV),

12

residual oil fly ash (n = 8, ROFA) and ROFA with RVS treatment (n = 8, ROFA + RSV).

13

The rats were exposed to ROFA by intranasal instillation and were treated with RVS

14

(20mg/kg/day) by gavage for 14 weeks. After twenty four hours, lung tissues were

15

collected for determination of oxidative damage markers (thiobarbituric acid reactive

16

substances - TBARS), antioxidant status (superoxide dismutase - SOD and catalase - CAT

17

activity), DNA damage, cytokines and metals levels. The results show no statistically

18

significant difference in TBARS, SOD and CAT activity, cytokines and metals levels

19

between groups. The ROFA group showed higher DNA damage when compared to the

20

other groups. In conclusion, the present study demonstrated that RSV avoided DNA

21

damage after subchronic ROFA exposure.

22 23 24

Keywords: resveratrol, air polluiton, ROFA, DNA damage, oxidative stress, inflammation, rat lung.

39 25

INTRODUCTION

26

27

Ambient air in polluted areas contains thousands of chemical compounds known to

28

possess mutagenic and carcinogenic properties. Many studies associate urban air pollution

29

with significant health effects on exposed population, including morbidity and mortality

30

due to cardiopulmonary diseases or lung cancer (Dominici et al., 2006; Fajersztajn et al.,

31

2013).

32

Among the air pollutants, particulate matter (PM) seems to be of major concern

33

from the health perspective (WHO, 2005). It is constituted by organic and inorganic

34

compounds. The inorganic residues, such as fly ashes, are the result of an incomplete

35

oxidation of carbonaceous materials and this pollutant significantly contributes to the

36

ambient air particulate burden. Because of the unique composition of the Residual Oil Fly

37

Ashes (ROFA), especially considering metals, it has been useful as surrogate for ambient

38

air PM exposure in many biological studies (Ghio et al., 2002). Previous data suggests that

39

ROFA administration via intratracheal/intranasal instillation and aerosol inhalation disclose

40

functional and structural alterations such as acute lung injury, alveolar septal thickening,

41

increased cellularity and lung inflammation (Mangnani et al., 2011 Ghio et al., 2002).

42

Resveratrol (3,5,4’-trihydroxystilbene; RSV) is a natural polyphenol found in some

43

plants, including grapes and their derivatives, berries and nuts. Recently, resveratrol has

44

received increasing attention because it has varied bioactivities, including the inhibition of

45

tumorigenesis, lipid modification, anti-inflammation, antioxidation and calorie-restriction

46

(reviewed by Park et al., 2015). Moreover, previous experiments have demonstrated that

47

resveratrol ameliorates trachea histological changes, lung inflammation and oxidative stress

48

in animal models of cigarette smoke (Kurus et al., 2009; Liu et al., 2014).

40 49

A comprehensive analysis of the time course of the changes in lung markers of

50

oxidative stress, inflammation and DNA damage after chronic ROFA exposure as well as

51

the potential protective effects of RSV have not yet been performed. Thus, we aimed to

52

investigate the subchronic exposure to ROFA and the effects of RSV intake on rat lungs.

53

54

MATERIALS AND METHODS

55

Animals

56

Male Wistar rats were obtained from Universidade Federal de Ciências da Saúde de

57

Porto Alegre (UFCSPA). Animals were maintained at controlled temperature (21±2ºC)

58

with 12/12- hour light/dark cycle and food and water ad libitum. All procedures were in

59

accordance with the Guide for the Care and Use of Laboratory Animals adopted by

60

National Institute of Health (NIH-USA). The study was approved by the Ethics Committee

61

of UFCSPA (Protocol 13-109) and a total of 33 rats were utilized.

62

63

Experimental design

64

ROFA suspension. ROFA was employed as a recognized ambient PM. ROFA

65

particles were collected from electrostatic precipitator installed in one of the chimneys of a

66

large steel plant in São Paulo, Brazil. Particles were prepared by suspending 20 μg of

67

ROFA particles in 10 μl sterile saline solution, and sonicated for 20 min in an ultrasonic

68

water bath.

69

70

RSV solution. RSV (≥ 98%, Pharma Nostra) was dissolved in saline with 0.05% of Tween 80.

71

Thirty-day-old male Wistar rats (200-283 g) were randomly distributed into four

72

groups: control (n=9; CTL); resveratrol (n=8; RSV); residual oil fly ash (n=8; ROFA); and

41 73

ROFA with RVS treatment (n=8; RSV+ROFA). Rats received daily 20 mg/kg of

74

resveratrol by oral gavage and 20 μg ROFA by intranasal instillation. The control groups

75

received only the vehicle.

76

Animals were euthanized after 14 weeks of treatment and lung was dissected. The

77

upper lobes and the inferior part of the right lung were used for oxidative stress and metal

78

analyses, respectively, and stored in – 80ºC. The inferior part of left lung was fixed in

79

formaldehyde for 48 hours and then embedded in paraffin.

80

81

Biochemical analyses

82

Tissue preparation

83

Lung tissue samples were homogenized in ice-cold 20 mM sodium phosphate

84

buffer, pH 7.4 (1:5, w/v), containing 120 mM KCl, and protein inhibitors (1 μg/mL

85

pepstatin, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 0.5 mM PMSF) with a Potter-

86

Elvehjem glass homogenizer. The obtained suspension was centrifuged at 600 g for 10 min

87

at 4 °C to remove nuclei and cell debris. The pellet was discarded and the supernatant was

88

used as tissue homogenate and kept at −80 °C until analysis.

89

90

91

92

Protein content Protein concentration of lung homogenates was measured by Bradford’s method (Bradford, 1976) using bovine serum albumin as standard.

93

94

Thiobarbituric acid reactive substances assay

95

Oxidative damage was determined as thiobarbituric acid reactive substances

96

(TBARS) using a fluorometric assay (Buege and Aust, 1978). Briefly, lung homogenates

42 97

were mixed with 10% (w/v) trichloroacetic acid and centrifuged to precipitate proteins. The

98

supernatants were added to thiobarbituric acid 0.67% (w/v) and incubated for 30 min at

99

100ºC. TBARS were extracted using butanol (1:1;v/v) and measured at 535 nm. The

100

amount of TBARS was expressed in nmol/mg of protein using ε = 1.56 105 M-1cm-1.

101

102

Superoxide dismutase activity

103

The superoxide dismutase (SOD) activity assay is based on the capacity of

104

pyrogallol to autoxidize (Marklund and Marklund 1984). The inhibition of auto-oxidation

105

of this compound occurs in the presence of SOD, whose activity was then indirectly

106

assayed at 420 nm. One unit of SOD is defined as the enzyme quantity capable of inhibiting

107

50% of the reaction. A calibration curve was performed with purified SOD as standard. The

108

results were expressed as USOD/mg protein.

109

110

Catalase activity

111

The catalase (CAT) activity was performed according to Aebi (1984). The reaction

112

mixture contained 33 mM H2O2 in 50 mM phosphate buffer (pH 7.0) and lung

113

homogeneized. The decomposition of hydrogen peroxide by CAT was determined at 25°C

114

at 240 nm for 120 sec. The results were expressed in pmol/mg protein.

115

116

Metal analysis

117

The analyses of total amount of chemical elements were carried out based on a

118

previously published method (Batista et al,. 2009). Prior to analysis, samples were

119

solubilized in 1 ml tetramethylammonium hydroxide (TMAH) 50% (v/v) at room

120

temperature for 12 h. Then, the volume was made up to 10 ml with a diluent containing

43 121

0.5% (v/v) HNO3 and 0.01% (v/v) Triton X-100. In all experiments 10 μg/L of the internal

122

standard rhodium (Rh) was used. Samples were directly analyzed by an inductively coupled

123

plasma mass spectrometer (DRC-ICP-MS ELAN DRCII, PerkinElmer, SCIEX, Norwalk,

124

USA) operating with high purity argon (99.999%, Praxaair, Brazil). In order to check the

125

accuracy of the analysis, certified reference materials SRM 1577c and 1577b (bovine liver)

126

from the National Institute of Standards and Technology (NIST) and SRM TORT-2

127

(Lobster Hepatopancreas) from European Virtual Institute for Speciation Analysis (EVISA)

128

were

analyzed

in

each

batch

of

ordinary

sample

analysis.

129

130

Comet Assay

131

The alkaline comet assay in lung cells was performed as described in the literature

132

with minor changes (Singh et al., 1988). Briefly, it was made a cell suspension of the lung

133

in PBS buffer (pH = 7.40) and 20 µL of cell suspensions were then rapidly embedded in 90

134

µL of 0.75% low-melting point agarose at 37 °C. After solidification, the slides were

135

immersed in iced-cold lysis solution (2.5 M NaCl, 100 mM EDTA and 10 mM Tris, pH

136

10.0; containing freshly added 1% (v/v) Triton X-100 and 10% (v/v) dimethylsulfoxide

137

(DMSO) at 4 °C in dark for a minimum of 1 h. Afterwards, to allow DNA unwinding,

138

slides were incubated in a freshly made alkaline electrophoresis buffer (0.3 M NaOH and 1

139

mM EDTA; pH > 13) at 4 ºC for 5 min in a horizontal electrophoresis box. The alkaline

140

electrophoresis was carried out for 15 min at 25 V and 300 mA. After electrophoresis,

141

slides were washed three times in a neutralization buffer (0.4 M Tris; pH 7.5) for 5 min and

142

left to dry overnight at room temperature. Then, the slides were fixed for 10 min in

143

trichloroacetic acid 15% (w/v), zinc sulfate 5% (w/v), glycerol 5% (v/v). Finally, the slides

144

were stained with silver nitrate (sodium carbonate 5% (w/v), ammonium nitrate 0.05%

44 145

(w/v), silver nitrate 0.05% (w/v), tungstosilicic acid 0.125% (w/v), formaldehyde 0.075%

146

(w/v), freshly prepared in the dark) (Garcia et al., 2007). The images (50-60 cells/slide)

147

were captured with high performance Nikon camera. The quantification of the DNA strand

148

breaks of the stored images was done using the CASP software (CASPLab®) by which

149

percentage of tail DNA, tail moment and olive tail moment were measurement (Końca et

150

al., 2003).

151

152

Immunohistochemistry

153

For immunohistochemical analysis, the lung sections were deparaffinized and

154

hydrated, endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide and

155

antigen was retrieved with trypsin or high temperature. After, slides were incubated with

156

primary antibodies anti-IL-6 (goat polyclonal antibody, 1:7000 dilution, Cat # sc-1265,

157

Santa Cruz, CA, USA) and anti-TNFα (goat polyclonal antibody, 1:7000 dilution, Cat # sc-

158

1350, Santa Cruz, CA, USA) for 1 h at 37°C in a moist chamber. Then, the slides were

159

washed in PBS and incubated with Vectastain ABC kit (Vector Elite, Vector Laboratories,

160

Inc.Ingold Road Burlingame, CA). After this step, the slides were washed in PBS and

161

followed by the revelation 3.3 Diaminobenzidine (DAB) chromogen (Sigma Chemical Co.,

162

St. Louis, MO, USA). Finally, the slides were counterstaining with Harry's hematoxylin

163

and mounted with cover slips. The software Image-Pro® Plus versão 4.5 (Media

164

Cybernetics – Silver Spring MD, USA) was used to measure the total area of analyzed lung

165

tissue and the positively marked tissue. The results are expressed in proportion of positively

166

stained tissue area per total lung tissue area.

167

168

45 169

170

Statistical analyses

171

Data are presented as mean ± standard deviation (SD). All analyses were performed

172

using SPSS software, version 15.0 (SPSS Inc., Chicago, IL, USA). The normality of the

173

data was tested by Kolmogorov-Smirnov. A one-way analysis of variance (ANOVA) and

174

Kruskal-Wallis was used to parametric and non-parametric data, respectively, followed by

175

post-hoc Bonferroni test. The significance level was set at 5%.

176

177

RESULTS

178

Metals concentrations

179

The concentration of chemical elements such as of copper (Cu), cadmium (Cd),

180

nickel (Ni), zinc (Zn), aluminium (Al), iron (Fe) in ROFA are described in Table 1. No

181

differences were observed among groups considering the levels of chemical elements in the

182

rat lungs (Table 2).

183

Table 1. Concentrations of chemical elements in ROFA.

Metal Pb Al Zn Cd Ba Cu Ni As Se Mn Sr Sb Fe Mg P Cr

µg/g (mean ± SD) 3.1 ± 0.09 789.9 ± 23.28 20.3 ± 0.04 0.04 ± 0.002 30.2 ± 0.31 9.7 ± 0.15 287.0 ± 10.8 4.1 ± 0.05 7.5 ± 0.20 48.3 ± 0.98 8.4 ± 0.16 2.3 ± 0.57 20,397.2 ± 283.3 372.5 ± 1.93 388.5 ± 255.8 7.6 ± 0.23

46 184

ROFA, residual oil fly ash. Values are mean ± SD of three determinations.

185 186 187 188 189

Table 2. Concentrations of chemical elements in the lungs from rats after 14 weeks of exposure.

Metal CTL RSV ROFA Cu 6.92 ± 0.36 6.93 ± 0.20 7.33 ± 0.82 Se 2.46 ± 0.20 2.63 ± 0.22 2.51 ± 0.24 Zn 60.34 ± 3.67 59.46 ± 3.65 62.24 ± 4.19 Mn 1.73 ± 0.57 1.55 ± 0.32 1.60 ± 0.58 Fe 1,196.36 ± 177.39 1,106.59 ± 160.26 1,157.85 ± 177.94 Mg 703.48 ± 61.70 701.85 ± 68.87 753.60 ± 53.98 P 11,358.37 ± 867.40 11,410.72 ± 1089.90 12,162.04 ± 846.17 Cr 4.99 ± 0.20 5.52 ± 0.28 5.11 ± 0.43 Pb 0.03 ± 0.01 0.03 ± 0.01 0.03 ± 0.02 Al 0.80 ± 0.34 0.67± 0.27 0.61 ± 0.11 Cd 0.01 ± 0.001 0.01 ± 0.003 0.01 ± 0.002 Ni 0.04 ± 0.01 0.04 ± 0.01 0.06 ± 0.02 As 4.48 ± 0.95 5.42 ± 1.69 3.81 ± 0.74 Sr 0.46 ± 0.10 0.45 ± 0.13 0.49 ± 0.07 Sb 0.03 ± 0.01 0.03 ± 0.01 0.02 ± 0.01 190 Data are expressed as mean ± SD. All values are in µg/g. 191 CTL, control; RSV, resveratrol; ROFA, Residual oil fly ash. 192 P > 0.05 (One-Way ANOVA).

ROFA+RSV 6.88 ± 0.68 2.50 ± 0.13 58.91 ± 7.52 1.64 ± 0.38 1,152.27 ± 189.30 671.85 ± 92.97 10,880.63 ± 1604.43 4.82 ± 0.23 0.04 ± 0.02 0.65 ± 0.12 0.01 ± 0.004 0.05 ± 0.01 3.88 ± 0.67 0.48 ± 0.06 0.03 ± 0.02

193

194

195

196

Oxidative markers No changes were observed in TBARS content (P=0.209) and in antioxidant status of CAT (P=0.421) and SOD (P=0.964) (Table 3).

197

198

Table 3. Measurement of oxidative stress in lungs from rats after 14 weeks of exposure. TBARS (nmol/mg protein) CAT (pmol/mg protein) SOD (USOD/mg protein)

199 200 201 202

CTL

RSV

ROFA

ROFA+RSV

5.49 ± 1.63 3.92 ± 0.67 0.25 ± 0.04

5.21 ± 2.52 3.20 ± 1.19 0.24 ± 0.47

5.14 ± 3.47 3.04 ± 1.59 0.24 ± 0.11

6.36 ± 1.54 3.88 ± 1.20 0.26 ± 0.04

Values are presented in mean ± SD. CTL, control; RSV, resveratrol; ROFA, residual oil fly ash. P > 0.05 (one way ANOVA).

47 203

204

205

206

Immunohistochemistry There were no statistically significant differences in IL-6 and TNF-α levels in the lung among groups (P=0.483; P=0.106, respectively) (data not shown).

207

208

DNA damage of lung cells

209

After 14 weeks of ROFA exposure and RSV treatment, comet assay was performed

210

to detect the degree of DNA damage in pulmonary cells. As shown in figure 1, tail DNA%,

211

tail length and Olive tail moment of lung cells in the ROFA exposed group were higher

212

than the others groups (P<0.001).

213

48

214 215

Figure 1. DNA damage in lung cells of rats (n= 8-9 per group) to ROFA and RSV

216

treatment for 14 weeks. CTL, control; RSV, resveratrol; ROFA, Residual oil fly ash. The

217

data are expressed as mean ± SD. *P < 0.001; One-Way ANOVA followed by Bonferroni

218

test.

219

49 220

DISCUSSION

221

In the present study, we employed in vivo subchronic exposure to better evaluate the

222

role of RSV against ROFA-induced pulmonary injury. For such purpose, oxidative stress,

223

inflammatory markers and DNA damage in lungs were used as markers. After subchronic

224

treatment we did not identify significant difference on oxidative and antioxidant markers,

225

inflammation and metal content among the groups. However, RSV prevented DNA damage

226

induced by ROFA.

227

ROFA is a suspension of the material produced after oil burning that has been used

228

in some experimental models aiming to elucidate the adverse health effects of air pollution

229

exposure (Arantes-Costa et al., 2008; Ghio et al., 2002; Marchini et al., 2014). Although

230

the use of this surrogate does not exactly mimic the overall exposure to air pollution, this

231

PM contains many components of air pollution such as metals. The ROFA used in this

232

work contains predominantly iron and aluminum as shown in table 1.

233

Different studies evaluated the role of oxidative stress, apoptosis, antioxidants,

234

inflammation and defects of lung repair mechanisms leading to tissue damage following air

235

pollution exposure (Mazzoli-Rocha at al., 2014; Magnani et al., 2011; Zin et al., 2012). Time

236

course studies have shown that functional and histological impairment of lung induced by

237

ROFA returned to control values 120 h after exposure (Carvalho et al., 2014). According to

238

Magnani et al., (2011), the macromolecular damage in lungs after acute ROFA exposure

239

occur at different induction times. This evidence is consistent with different susceptibility

240

to oxidative damage as related by previous study (Gurgueira et al., 2002).

241

To our knowledge, this is the first study that evaluated the presence of chemical

242

elements in lungs after subchronic ROFA exposure. Wallenborn et al., (2007), observed

243

that PM-associate metals can translocate to systemic circulation and extrapulmonary organs

50 244

following a single intratracheal instillation based on their solubility; the less water-soluble

245

metals are retained in the lung longer. This suggests a mechanism of rapid removal via

246

either pulmonary capillaries or lung associated lymph nodes into the blood stream along

247

with mucociliary clearance.

248

The same research group demonstrated that soluble zinc introduced through the

249

lungs not only reaches but also accumulates in the heart following pulmonary exposure

250

(Wallenborn et al., 2009). More specifically, soluble components present in PM, including

251

metals, may translocate outside of the lung and reach extrapulmonary organs after a single

252

dose exposure. Oberdörster et al., (2004) have shown that particles can reach the central

253

nervous system (CNS) by nasal uptake but unlike lungs, the CNS does not have efficient

254

mechanisms of clearance. Based on our results - that have shown no difference in metal

255

levels in lungs - we suggest that metals could have been accumulated on extrapulmonary

256

organs after chronic exposure, although specific organs analyses were not done.

257

Oxidative stress and inflammation has been reported as a consequence to short-term

258

inhalation exposure to concentrated ambient particles (Rhoden et al., 2004). ROFA

259

produced oxidative stress in rat lungs after acute exposure (data not published), however

260

this mechanism of damage was not reported regarding chronic exposure (Damiani et al.,

261

2012) using the same exposure time but a different range of ROFA concentrations.

262

The majority of the studies have analyzed the adverse effects in acute exposures

263

while few have investigating effects in subchronic and chronic exposure to air pollution.

264

Responses to ROFA may vary depending on the composition of soluble metals, their

265

interaction with each other and with the cells, the concentration used as well as the time of

266

exposure.

51 267

RSV was employed in this study as an antioxidant with potential benefits. We

268

choose the oral administration since their route usually occur considering food containing

269

the compound. RSV has been reported to exert potent anti-oxidant activities not only

270

directly by scavenging hydroxyl, superoxide and metal-induced radicals (Bradamante et al.,

271

2004), but also indirectly by enhancing the activity of anti-oxidant enzymes such as

272

catalase and glutathione peroxidase (Spanier et al., 2009) and decreasing H2O2 and MDA

273

levels (Zhang et al., 2014). For instance, RSV has been reported to protect against cigarette

274

smoke-mediated oxidative stress in human lung epithelial cells (Kode et al., 2008) and

275

endotoxemia associated lung tissue injury in mice (Zhang et al., 2014).

276

Other compounds such as PM, organic extracts from PM, diesel exhausted particles,

277

coal fly ash, has been associated with DNA damage in cells (Topinka et al., 2012; Sharma

278

et al., 2007; Gabelová et al., 2007). Different studies have investigated the effects of air

279

pollution exposure on DNA damage in vivo. Meng et al., (2007) observed an increase of

280

DNA damage in rat lung cells in a dose–response manner due PM2.5 exposure.

281

Additionally, DEP caused oxidative DNA damage in guinea pigs lungs (Moller et al.,

282

2003).

283

In order to investigate the overall genotoxicity produced by the different chemical

284

components of ROFA we used the alkaline version of the Comet assay, which is sensitive

285

to detect DNA strand breaks, oxidative DNA lesions, and alkali-labile sites and has been

286

proposed as a useful tool for assessing the genotoxicity of particles (Singh et al., 1988;

287

Schins 2002). Interesting findings are reported concerning the genotoxic properties like the

288

role of organic extractable mutagens such as PAHs (Hsiao et al., 2000) and the genotoxic

289

effects of transition metals due its ability to generate ROS (Prahalad et al., 2001; Knaapen

290

et al., 2002). In our study we did not find differences in oxidative stress and inflammation,

52 291

what suggest that DNA damage were caused directly by the particles and the RVT

292

treatment avoided the damage induced by ROFA (Fig. 1). Besides, it is important to

293

emphasize that cells have DNA protection systems in tumor prevention, such nucleotide-

294

excision repair (NER), base-excision repair (BER), which could avoid irreversible

295

mutations that contribute to oncogenesis (Hoeijmakers, 2001).

296

Many in vitro and in vivo animal models have demonstrated the potent protection

297

conferred by RSV against inflammation, oxidative stress, and cancer (Baur and Sinclair,

298

2006). RSV can promote cell cycle arrest leading to apoptosis of tumor cells, prevent

299

tumor-derived nitric oxide synthase expression to block tumor growth and migration, as

300

well as act as an antioxidant to prevent DNA damage that can lead to tumor formation

301

(Clément et al. 1998; Spanier et al., 2009; Park et al., 2015).

302

This study presents limitations. Ideally if the animals were exposed to

303

environmental air rather than to intranasal suspensions of ROFA more precise outcomes

304

determined by the ambient air could be found. Secondly, we did not measure the content of

305

PAHs and other constituents of ROFA, which could explain some findings.

306

In conclusion, this work provides new insights for understanding the mechanisms

307

involved in lung damage due to subchronic exposure to ambient particles for 14 weeks and

308

the beneficial effects of RSV treatment.

309

310

CONCLUSIONS

311

This is the first study to demonstrate that RSV could protect DNA damage due

312

ROFA exposure. However, we emphasize further studies are needed to clarify whether the

313

metals accumulates in others organs as well as which ROFA components are associated

314

with the outcomes.

53 315

316

ACKNOWLEDGEMENTS

317

This work was funded by CAPES, FAPESP, CNPq, and by the Universidade

318

Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil and

319

Faculdade de Medicina de São Paulo, São Paulo, Brazil.

54

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57

6 CONSIDERAÇÕES FINAIS

Segundo os resultados obtidos neste trabalho, podemos concluir que a exposição subcrônica de ratos ao ROFA e ao RSV:

1) Não altera os danos oxidativos no pulmão; 2) Não altera a atividade das enzimas antioxidantes CAT e SOD no pulmão; 3) Não altera a concentração de citocinas pro-inflamatórias IL-6, TNF-α no tecido pulmonar; 4) Não altera a concentração de metais no pulmão; 5) Altera o dano ao DNA das células pulmonares, sendo que o ROFA danifica e o RSV previne esse tipo de dano.

Sendo assim, o RSV foi capaz de prevenir o dano ao DNA causado pela exposição ao ROFA, entretanto não foi observada alteração de resposta pulmonar nos demais parâmetros analisados. Esse estudo sugere que o pulmão é capaz de se adaptar as condições ambientais após exposição subcrônica ao ROFA e, por isso, não observamos modificações quanto aos parâmetros analisados de estresse oxidativo, assim como a concentração de interleucinas pró-inflamatórias e de elementos químicos. Contudo, verificou-se que o RSV foi capaz de prevenir o dano ao DNA das células pulmonares causado pela exposição ao ROFA. Devido à complexidade dos mecanismos de defesa celular e ao protocolo empregado, não é possível afirmar que tais danos ao DNA sejam capazes de desencadear carcinogênese. Por isso, mais estudos são necessários para investigar os mecanismos de defesa pulmonar as partículas ambientais a longo prazo, além dos mecanismos de proteção contra o dano ao DNA estabelecido pelo RVS.

58

ANEXO A – PARECER DA COMISSÃO DE ÉTICA NO USO DE ANIMAIS (CEUA)

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ANEXO B – NORMAS DE PUBLICAÇÃO DA REVISTA

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Changes to authorship This policy concerns the addition, deletion, or rearrangement of author names in the authorship of accepted manuscripts: Before the accepted manuscript is published in an online issue: Requests to add or remove an author, or to rearrange the author names, must be sent to the Journal Manager from the corresponding author of the accepted manuscript and must include: (a) the reason the name should be added or removed, or the author names rearranged and (b) written confirmation (e-mail, fax, letter) from all authors that they agree with the addition, removal or rearrangement. In the case of addition or removal of authors, this includes confirmation from the author being added or removed. Requests that are not sent by the corresponding author will be forwarded by the Journal Manager to the corresponding author, who must follow the procedure as described above. Note that: (1) Journal Managers will inform the Journal Editors of any such requests and (2) publication of the accepted manuscript in an online issue is suspended until authorship has been agreed. After the accepted manuscript is published in an online issue: Any requests to add, delete, or rearrange author names in an article published in an online issue will follow the same policies as noted above and result in a corrigendum.

Article transfer service This journal is part of our Article Transfer Service. This means that if the Editor feels your article is more suitable in one of our other participating journals, then you may be asked to consider transferring the article to one of those. If you agree, your article will be transferred automatically on your behalf with no need to reformat. Please note that your article will be reviewed again by the new journal. More information about this can be found here: http://www.elsevier.com/authors/article-transfer-service.

Copyright Upon acceptance of an article, authors will be asked to complete a 'Journal Publishing Agreement' (for more information on this and copyright, see http://www.elsevier.com/copyright). An e-mail will be sent to the corresponding author confirming receipt of the manuscript together with a 'Journal Publishing Agreement' form or a link to the online version of this agreement.

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Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal circulation within their institutions. Permission of the Publisher is required for resale or distribution outside the institution and for all other derivative works, including compilations and translations (please consult http://www.elsevier.com/permissions). If excerpts from other copyrighted works are included, the author(s) must obtain written permission from the copyright owners and credit the source(s) in the article. Elsevier has preprinted forms for use by authors in these cases: please consult http://www.elsevier.com/permissions. For open access articles: Upon acceptance of an article, authors will be asked to complete an 'Exclusive License Agreement' (for more information see http://www.elsevier.com/OAauthoragreement). Permitted third party reuse of open access articles is determined by the author's choice of user license (see http://www.elsevier.com/openaccesslicenses). Author rights As an author you (or your employer or institution) have certain rights to reuse your work. For more information see http://www.elsevier.com/copyright.

Role of the funding source You are requested to identify who provided financial support for the conduct of the research and/or preparation of the article and to briefly describe the role of the sponsor(s), if any, in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. If the funding source(s) had no such involvement then this should be stated.

Funding body agreements and policies Elsevier has established a number of agreements with funding bodies which allow authors to comply with their funder's open access policies. Some authors may also be reimbursed for associated publication fees. To learn more about existing agreements please visit http://www.elsevier.com/fundingbodies. US National Institutes of Health (NIH) voluntary posting (" Public Access") policy. Elsevier facilitates author response to the NIH voluntary posting request (referred to as the NIH "Public Access Policy"; see http://www.nih.gov/about/publicaccess/index.htm)by posting the peer reviewed author's manuscript directly to PubMed Central on request from the author, 12 months after formal publication. Upon notification from Elsevier of acceptance, we will ask you to confirm via email (by e-mailing us at [email protected] ) that your work has received NIH funding and that you intend to respond to the NIH policy request, along with your NIH award number to facilitate processing. Upon such confirmation, Elsevier will submit to PubMed Central on your behalf a version of your manuscript that will include peer-review comments, for posting 12 months after formal publication. This will ensure that you will have responded fully to the NIH request policy. There will be no need for you to post your manuscript directly with PubMed Central, and any such posting is prohibited.

Open access This journal offers authors a choice in publishing their research: Open access • Articles are freely available to both subscribers and the wider public with permitted reuse • An open access publication fee is payable by authors or on their behalf e.g. by their research funder or institution Subscription

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• Articles are made available to subscribers as well as developing countries and patient groups through our universal access programs (http://www.elsevier.com/access). • No open access publication fee payable by authors. Regardless of how you choose to publish your article, the journal will apply the same peer review criteria and acceptance standards. For open access articles, permitted third party (re)use is defined by the following Creative Commons user licenses: Creative Commons Attribution (CC BY) Lets others distribute and copy the article, create extracts, abstracts, and other revised versions, adaptations or derivative works of or from an article (such as a translation), include in a collective work (such as an anthology), text or data mine the article, even for commercial purposes, as long as they credit the author(s), do not represent the author as endorsing their adaptation of the article, and do not modify the article in such a way as to damage the author's honor or reputation. Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) For non-commercial purposes, lets others distribute and copy the article, and to include in a collective work (such as an anthology), as long as they credit the author(s) and provided they do not alter or modify the article. The open access publication fee for this journal is USD 3000, excluding taxes. Learn more about Elsevier's pricing policy: http://www.elsevier.com/openaccesspricing.

Language (usage and editing services) Please write your text in good English (American or British usage is accepted, but not a mixture of these). Authors who feel their English language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientific English may wish to use the English Language Editing service available from Elsevier's WebShop (http://webshop.elsevier.com/languageediting/) or visit our customer support site (http://support.elsevier.com) for more information.

Submission Our online submission system guides you stepwise through the process of entering your article details and uploading your files. The system converts your article files to a single PDF file used in the peer-review process. Editable files (e.g., Word, LaTeX) are required to typeset your article for final publication. All correspondence, including notification of the Editor's decision and requests for revision, is sent by e-mail. Should you be unable to provide an electronic version, please contact the editorial office prior to submission at e-mail: [email protected]; telephone: +1 (619) 699-6275; or fax: +1 (619) 699-6211.

Experimental procedures All animal experiments should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, the European Communities Council Directive of 24 November 1986 (86/609/EEC) or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and the authors should clearly indicate in the manuscript that such guidelines have been followed. All animal studies need to ensure they comply with the ARRIVE guidelines. More information can be found at http://www.nc3rs.org.uk/page.asp?id=1357.

PREPARATION NEW SUBMISSIONS

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Submission to this journal proceeds totally online and you will be guided stepwise through the creation and uploading of your files. The system automatically converts your files to a single PDF file, which is used in the peer-review process. As part of the Your Paper Your Way service, you may choose to submit your manuscript as a single file to be used in the refereeing process. This can be a PDF file or a Word document, in any format or layout that can be used by referees to evaluate your manuscript. It should contain high enough quality figures for refereeing. If you prefer to do so, you may still provide all or some of the source files at the initial submission. Please note that individual figure files larger than 10 MB must be uploaded separately.

References There are no strict requirements on reference formatting at submission. References can be in any style or format as long as the style is consistent. Where applicable, author(s) name(s), journal title/book title, chapter title/article title, year of publication, volume number/book chapter and the pagination must be present. Use of DOI is highly encouraged. The reference style used by the journal will be applied to the accepted article by Elsevier at the proof stage. Note that missing data will be highlighted at proof stage for the author to correct.

Formatting requirements There are no strict formatting requirements but all manuscripts must contain the essential elements needed to convey your manuscript, for example Abstract, Keywords, Introduction, Materials and Methods, Results, Conclusions, Artwork and Tables with Captions. If your article includes any Videos and/or other Supplementary material, this should be included in your initial submission for peer review purposes. Divide the article into clearly defined sections. Please ensure the text of your paper is double-spaced and has consecutive line numbering– this is an essential peer review requirement. Figures and tables embedded in text Please ensure the figures and the tables included in the single file are placed next to the relevant text in the manuscript, rather than at the bottom or the top of the file.

REVISED SUBMISSIONS Use of word processing software Regardless of the file format of the original submission, at revision you must provide us with an editable file of the entire article. Keep the layout of the text as simple as possible. Most formatting codes will be removed and replaced on processing the article. The electronic text should be prepared in a way very similar to that of conventional manuscripts (see also the Guide to Publishing with Elsevier: http://www.elsevier.com/guidepublication). See also the section on Electronic artwork. To avoid unnecessary errors you are strongly advised to use the 'spell-check' and 'grammar-check' functions of your word processor.

Article structure Preparation of Manuscript. Manuscripts text should have double line spacing and references should be single-spaced. Pages should be numbered consecutively and organized as follows: The title Page (p. 1) should contain the article title, authors' names and complete affiliations, footnotes to the title, and the address for manuscript correspondence (including e-mail address and telephone and fax numbers). The article title should be comprehensive and descriptive: proprietary names must not be used in titles, but may be identified in footnotes.

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Page 2 should contain an abstract. The abstract should be limited to 250 words but must contain a concise summary of what was done, the results obtained, and valid conclusions which are drawn therefrom. It must mention the compounds or families of compounds studied, their actions, and the species of animals. It must contain important words which are used as index terms, but not proprietary names. Keywords should be listed immediately after the abstract. Format. The following text sections should be used. Introduction. State why the investigation was carried out, note any relevant published work, and delineate the objective of the investigation. Methods. New methods or significant improvements of methods or changes in old methods must be described. Methods for which adequate reference can be cited are not to be described. In the Methods section, authors should draw attention to any particular chemical or biological hazards that may be involved in carrying out the experiments described. Any relevant safety precautions should be described: if an accepted code of practice has been followed, a reference to the relevant standards should be given. Details regarding animal housing conditions should be given. Results. Duplication between the text of this section and material presented in tables and figures should be avoided. Tabular presentation of masses of negative data must be avoided and replaced with a statement in the text whenever possible. The statement must include (a) what was done, (b) how it was done, (c) how the data were analyzed, (d) a measure of variability, and (e) the significance of the result. Discussion. This section must relate to the significance of the work to existing knowledge in the field and indicate the importance of the contribution of this study. Needless detailed recapitulation of the results must be avoided. Unsupported hypotheses and speculation should be omitted. Subdivision - unnumbered sections Divide your article into clearly defined sections. Each subsection is given a brief heading. Each heading should appear on its own separate line. Subsections should be used as much as possible when crossreferencing text: refer to the subsection by heading as opposed to simply 'the text'. Introduction State the objectives of the work and provide an adequate background, avoiding a detailed literature survey or a summary of the results. Material and methods Provide sufficient detail to allow the work to be reproduced. Methods already published should be indicated by a reference: only relevant modifications should be described. Authors should draw attention to any particular chemical or biological hazards that may be involved in carrying out the experiments described. Any relevant safety precautions should be described: if an accepted code of practice has been followed, a reference to the relevant standards should be given. Details regarding animal housing should also be notated. Results Results should be clear and concise. Duplication between the text of this section and material presented in tables and figures should be avoided and replaced with a statement in the text whenever possible. The statement must include (a) what was done, (b) how it was done, (c ) how the data were analyzed, (d) a measure of variability, and (e) the significance of the result.

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Discussion This should explore the significance of the results of the work, not repeat them. A combined Results and Discussion section is sometmes appropriate. Avoid extensive citations and discussion of published literature. Needless detailed recapitulation of the results must be avoided. Unsupported hypotheses and speculation should also be omitted. Appendices If there is more than one appendix, they should be identified as A, B, etc. Formulae and equations in appendices should be given separate numbering: Eq. (A.1), Eq. (A.2), etc.; in a subsequent appendix, Eq. (B.1) and so on. Similarly for tables and figures: Table A.1; Fig. A.1, etc.

Essential title page information

• Title. Concise and informative. Titles are often used in information-retrieval systems. Avoid abbreviations and formulae where possible. • Author names and affiliations. Please clearly indicate the given name(s) and family name(s) of each author and check that all names are accurately spelled. Present the authors' affiliation addresses (where the actual work was done) below the names. Indicate all affiliations with a lowercase superscript letter immediately after the author's name and in front of the appropriate address. Provide the full postal address of each affiliation, including the country name and, if available, the e-mail address of each author. • Corresponding author. Clearly indicate who will handle correspondence at all stages of refereeing and publication, also post-publication. Ensure that the e-mail address is given and that contact details are kept up to date by the corresponding author. • Present/permanent address. If an author has moved since the work described in the article was done, or was visiting at the time, a 'Present address' (or 'Permanent address') may be indicated as a footnote to that author's name. The address at which the author actually did the work must be retained as the main, affiliation address. Superscript Arabic numerals are used for such footnotes.

Abstract A concise and factual abstract is required. The abstract should state briefly the purpose of the research, the principal results and major conclusions. An abstract is often presented separately from the article, so it must be able to stand alone. For this reason, References should be avoided, but if essential, then cite the author(s) and year(s). Also, non-standard or uncommon abbreviations should be avoided, but if essential they must be defined at their first mention in the abstract itself. The abstract should be limited to 250 words. It must mention the compounds or families of coumpounds studied, their actions, and the species of animals, but must not contain proprietary names.

Graphical abstract Although a graphical abstract is optional, its use is encouraged as it draws more attention to the online article. The graphical abstract should summarize the contents of the article in a concise, pictorial form designed to capture the attention of a wide readership. Graphical abstracts should be submitted as a separate file in the online submission system. Image size: Please provide an image with a minimum of 531 × 1328 pixels (h × w) or proportionally more. The image should be readable at a size of 5 × 13 cm using a regular screen resolution of 96 dpi. Preferred file types: TIFF, EPS, PDF or MS Office files. See http://www.elsevier.com/graphicalabstracts for examples.

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Authors can make use of Elsevier's Illustration and Enhancement service to ensure the best presentation of their images and in accordance with all technical requirements: Illustration Service.

Highlights Highlights are mandatory for this journal. They consist of a short collection of bullet points that convey the core findings of the article and should be submitted in a separate editable file in the online submission system. Please use 'Highlights' in the file name and include 3 to 5 bullet points (maximum 85 characters, including spaces, per bullet point). See http://www.elsevier.com/highlights for examples.

Keywords Immediately after the abstract, provide a maximum of 6 keywords, using American spelling and avoiding general and plural terms and multiple concepts (avoid, for example, 'and', 'of'). Be sparing with abbreviations: only abbreviations firmly established in the field may be eligible. These keywords will be used for indexing purposes.

Abbreviations Define abbreviations that are not standard in this field in a footnote to be placed on the first page of the article. Such abbreviations that are unavoidable in the abstract must be defined at their first mention there, as well as in the footnote. Ensure consistency of abbreviations throughout the article.

Acknowledgements Collate acknowledgements in a separate section at the end of the article before the references and do not, therefore, include them on the title page, as a footnote to the title or otherwise. List here those individuals who provided help during the research (e.g., providing language help, writing assistance or proof reading the article, etc.). Units will be in general accordance with the International System (SI) as adopted by the 11the General Conference on Weights and Measures. Common abbreviations to be used in this journal are: m meter ppm parts per million cm centimeter cpm counts per minute mm millimeter dpm disintegrations per minute um micrometer sc subcutaneous nm nanometer ic intracutaneous kg kilogram im intramuscular g gram ip intraperitoneal mg milligram iv intravenous ug microgram po oral ng nanogram LD50 medial lethal dose ml milliliter LC50 medial lethal concentration >ul microliter Hz hertz mol mole s seconds M molar

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min minutes mM millimolar h hours uM micromolar SD standard deviation N normal SE standard error Ci Curie TLV threshold limit value X mean

Database linking Elsevier encourages authors to connect articles with external databases, giving their readers oneclick access to relevant databases that help to build a better understanding of the described research. Please refer to relevant database identifiers using the following format in your article: Database: xxxx (e.g., TAIR: AT1G01020; CCDC: 734053; PDB: 1XFN). See http://www.elsevier.com/databaselinking for more information and a full list of supported databases.

Math formulae Please submit math equations as editable text and not as images. Present simple formulae in line with normal text where possible and use the solidus (/) instead of a horizontal line for small fractional terms, e.g., X/Y. In principle, variables are to be presented in italics. Powers of e are often more conveniently denoted by exp. Number consecutively any equations that have to be displayed separately from the text (if referred to explicitly in the text). Proprietary names of substances and names and addresses of suppliers should be identified in footnotes. If the paper has been presented orally in whole or part, the date, and occasion should be included in a footnote.

Footnotes Footnotes should be used sparingly. Number them consecutively throughout the article. Many word processors build footnotes into the text, and this feature may be used. Should this not be the case, indicate the position of footnotes in the text and present the footnotes themselves separately at the end of the article.

Artwork Image manipulation Whilst it is accepted that authors sometimes need to manipulate images for clarity, manipulation for purposes of deception or fraud will be seen as scientific ethical abuse and will be dealt with accordingly. For graphical images, this journal is applying the following policy: no specific feature within an image may be enhanced, obscured, moved, removed, or introduced. Adjustments of brightness, contrast, or color balance are acceptable if and as long as they do not obscure or eliminate any information present in the original. Nonlinear adjustments (e.g. changes to gamma settings) must be disclosed in the figure legend. Electronic artwork General points • Make sure you use uniform lettering and sizing of your original artwork. • Preferred fonts: Arial (or Helvetica), Times New Roman (or Times), Symbol, Courier. • Number the illustrations according to their sequence in the text. • Use a logical naming convention for your artwork files. • Indicate per figure if it is a single, 1.5 or 2-column fitting image. • For Word submissions only, you may still provide figures and their captions, and tables within a single file at the revision stage.

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• Please note that individual figure files larger than 10 MB must be provided in separate source files. A detailed guide on electronic artwork is available on our website: http://www.elsevier.com/artworkinstructions. You are urged to visit this site; some excerpts from the detailed information are given here. Formats Regardless of the application used, when your electronic artwork is finalized, please 'save as' or convert the images to one of the following formats (note the resolution requirements for line drawings, halftones, and line/halftone combinations given below): EPS (or PDF): Vector drawings. Embed the font or save the text as 'graphics'. TIFF (or JPG): Color or grayscale photographs (halftones): always use a minimum of 300 dpi. TIFF (or JPG): Bitmapped line drawings: use a minimum of 1000 dpi. TIFF (or JPG): Combinations bitmapped line/half-tone (color or grayscale): a minimum of 500 dpi is required. Please do not: • Supply files that are optimized for screen use (e.g., GIF, BMP, PICT, WPG); the resolution is too low. • Supply files that are too low in resolution. • Submit graphics that are disproportionately large for the content. Color artwork Please make sure that artwork files are in an acceptable format (TIFF (or JPEG), EPS (or PDF), or MS Office files) and with the correct resolution. If, together with your accepted article, you submit usable color figures then Elsevier will ensure, at no additional charge, that these figures will appear in color online (e.g., ScienceDirect and other sites) regardless of whether or not these illustrations are reproduced in color in the printed version. For color reproduction in print, you will receive information regarding the costs from Elsevier after receipt of your accepted article. Please indicate your preference for color: in print or online only. For further information on the preparation of electronic artwork, please see http://www.elsevier.com/artworkinstructions. Please note: Because of technical complications that can arise by converting color figures to 'gray scale' (for the printed version should you not opt for color in print) please submit in addition usable black and white versions of all the color illustrations. Figure captions Ensure that each illustration has a caption. A caption should comprise a brief title (not on the figure itself) and a description of the illustration. Keep text in the illustrations themselves to a minimum but explain all symbols and abbreviations used.

Tables Please submit tables as editable text and not as images. Tables can be placed either next to the relevant text in the article, or on separate page(s) at the end. Number tables consecutively in accordance with their appearance in the text and place any table notes below the table body. Be sparing in the use of tables and ensure that the data presented in them do not duplicate results described elsewhere in the article. Please avoid using vertical rules.

References Unpublished results or personal communications should be cited as such in the text. Citation in text Please ensure that every reference cited in the text is also present in the reference list (and vice versa). Any references cited in the abstract must be given in full. Unpublished results and personal communications are not recommended in the reference list, but may be mentioned in the text. If these references are included in the reference list they should follow the standard reference style of the journal and should include a substitution of the publication date with either 'Unpublished results' or 'Personal communication'.

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Citation of a reference as 'in press' implies that the item has been accepted for publication and a copy of the title page of the relevant article must be submitted. Reference links Increased discoverability of research and high quality peer review are ensured by online links to the sources cited. In order to allow us to create links to abstracting and indexing services, such as Scopus, CrossRef and PubMed, please ensure that data provided in the references are correct. Please note that incorrect surnames, journal/book titles, publication year and pagination may prevent link creation. When copying references, please be careful as they may already contain errors. Use of the DOI is encouraged. Web references As a minimum, the full URL should be given and the date when the reference was last accessed. Any further information, if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references can be listed separately (e.g., after the reference list) under a different heading if desired, or can be included in the reference list. References in a special issue Please ensure that the words 'this issue' are added to any references in the list (and any citations in the text) to other articles in the same Special Issue. Reference management software This journal has standard templates available in key reference management packages EndNote (http://www.endnote.com/support/enstyles.asp) and Reference Manager (http://refman.com/support/rmstyles.asp). Using plug-ins to wordprocessing packages, authors only need to select the appropriate journal template when preparing their article and the list of references and citations to these will be formatted according to the journal style which is described below. Reference formatting There are no strict requirements on reference formatting at submission. References can be in any style or format as long as the style is consistent. Where applicable, author(s) name(s), journal title/book title, chapter title/article title, year of publication, volume number/book chapter and the pagination must be present. Use of DOI is highly encouraged. The reference style used by the journal will be applied to the accepted article by Elsevier at the proof stage. Note that missing data will be highlighted at proof stage for the author to correct. If you do wish to format the references yourself they should be arranged according to the following examples: Reference style Text: All citations in the text should refer to: 1. Single author: the author's name (without initials, unless there is ambiguity) and the year of publication; 2. Two authors: both authors' names and the year of publication; 3. Three or more authors: first author's name followed by 'et al.' and the year of publication. Citations may be made directly (or parenthetically). Groups of references should be listed first alphabetically, then chronologically. Examples: 'as demonstrated (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999). Kramer et al. (2010) have recently shown ....' List: References should be arranged first alphabetically and then further sorted chronologically if necessary. More than one reference from the same author(s) in the same year must be identified by the letters 'a', 'b', 'c', etc., placed after the year of publication. Examples: Reference to a journal publication:

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Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2010. The art of writing a scientific article. J. Sci. Commun. 163, 51–59. Reference to a book: Strunk Jr., W., White, E.B., 2000. The Elements of Style, fourth ed. Longman, New York. Reference to a chapter in an edited book: Mettam, G.R., Adams, L.B., 2009. How to prepare an electronic version of your article, in: Jones, B.S., Smith , R.Z. (Eds.), Introduction to the Electronic Age. E-Publishing Inc., New York, pp. 281–304. Journal abbreviations source Journal names should be abbreviated according to the List of Title Word Abbreviations: http://www.issn.org/services/online-services/access-to-the-ltwa/.

Video data Elsevier accepts video material and animation sequences to support and enhance your scientific research. Authors who have video or animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article. This can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be placed. All submitted files should be properly labeled so that they directly relate to the video file's content. In order to ensure that your video or animation material is directly usable, please provide the files in one of our recommended file formats with a preferred maximum size of 50 MB. Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products, including ScienceDirect: http://www.sciencedirect.com. Please supply 'stills' with your files: you can choose any frame from the video or animation or make a separate image. These will be used instead of standard icons and will personalize the link to your video data. For more detailed instructions please visit our video instruction pages at http://www.elsevier.com/artworkinstructions. Note: since video and animation cannot be embedded in the print version of the journal, please provide text for both the electronic and the print version for the portions of the article that refer to this content.

AudioSlides The journal encourages authors to create an AudioSlides presentation with their published article. AudioSlides are brief, webinar-style presentations that are shown next to the online article on ScienceDirect. This gives authors the opportunity to summarize their research in their own words and to help readers understand what the paper is about. More information and examples are available at http://www.elsevier.com/audioslides. Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides presentation after acceptance of their paper.

Supplementary data Elsevier accepts electronic supplementary material to support and enhance your scientific research. Supplementary files offer the author additional possibilities to publish supporting applications, high resolution images, background datasets, sound clips and more. Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products, including ScienceDirect: http://www.sciencedirect.com. In order to ensure that your submitted material is directly usable, please provide the data in one of our recommended file formats. Authors should submit the material in electronic format together with the article and supply a concise and descriptive caption for each file. For more detailed instructions please visit our artwork instruction pages at http://www.elsevier.com/artworkinstructions. Alternatively, authors may convert any or all parts of their supplementary data into one or multiple Data in Brief articles, a new kind of article that houses and describes their data. Data in Brief articles ensure that your data, which is normally buried in

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supplementary material, is actively reviewed, curated, formatted, indexed, given a DOI and publicly available to all upon publication. You can submit your Data in Brief directly alongside your research article submission (either initially or at the revision stage). If your research article is accepted, your Data in Brief article will be editorially reviewed and published in the new, Open Access journal, Data in Brief. Your Data in Brief and research article will directly cite and link to each other (see published examples). The open access fees will be waived if your article is submitted by December 31, 2014. Please use the following template to write your Data in Brief."

Submission checklist The following list will be useful during the final checking of an article prior to sending it to the journal for review. Please consult this Guide for Authors for further details of any item. Ensure that the following items are present: One author has been designated as the corresponding author with contact details: • E-mail address • Full postal address All necessary files have been uploaded, and contain: • Keywords • All figure captions • All tables (including title, description, footnotes) Further considerations • Manuscript has been 'spell-checked' and 'grammar-checked' • All references mentioned in the Reference list are cited in the text, and vice versa • Permission has been obtained for use of copyrighted material from other sources (including the Internet) Printed version of figures (if applicable) in color or black-and-white • Indicate clearly whether or not color or black-and-white in print is required. • For reproduction in black-and-white, please supply black-and-white versions of the figures for printing purposes. For any further information please visit our customer support site at http://support.elsevier.com.

AFTER ACCEPTANCE Use of the Digital Object Identifier The Digital Object Identifier (DOI) may be used to cite and link to electronic documents. The DOI consists of a unique alpha-numeric character string which is assigned to a document by the publisher upon the initial electronic publication. The assigned DOI never changes. Therefore, it is an ideal medium for citing a document, particularly 'Articles in press' because they have not yet received their full bibliographic information. Example of a correctly given DOI (in URL format; here an article in the journal Physics Letters B): http://dx.doi.org/10.1016/j.physletb.2010.09.059 When you use a DOI to create links to documents on the web, the DOIs are guaranteed never to change.

Proofs One set of page proofs (as PDF files) will be sent by e-mail to the corresponding author (if we do not have an e-mail address then paper proofs will be sent by post) or, a link will be provided in the e-mail so that authors can download the files themselves. Elsevier now provides authors with PDF proofs which can be annotated; for this you will need to download Adobe Reader version 9 (or higher) available free from http://get.adobe.com/reader. Instructions on how to annotate PDF files will accompany the proofs (also given online). The exact system requirements are given at the Adobe site: http://www.adobe.com/products/reader/tech-specs.html. If you do not wish to use the PDF annotations function, you may list the corrections (including replies to the Query Form) and return them to Elsevier in an e-mail. Please list

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your corrections quoting line number. If, for any reason, this is not possible, then mark the corrections and any other comments (including replies to the Query Form) on a printout of your proof and return by fax, or scan the pages and e-mail, or by post. Please use this proof only for checking the typesetting, editing, completeness and correctness of the text, tables and figures. Significant changes to the article as accepted for publication will only be considered at this stage with permission from the Editor. We will do everything possible to get your article published quickly and accurately. It is important to ensure that all corrections are sent back to us in one communication: please check carefully before replying, as inclusion of any subsequent corrections cannot be guaranteed. Proofreading is solely your responsibility.

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Efeitos da exposição subcrônica de Residual Oil Fly Ash (ROFA

UNIVERSIDADE FEDERAL DE CIÊNCIAS DA SAÚDE DE PORTO ALEGRE – UFCSPA CURSO DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE Marlise Di Domenico Efeitos da exposi...

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