Beatriz Aparecida Fioruci Fontanelli
"Efeitos do consumo crônico de etanol sobre a atividade
de MMP-2 / MMP-9 e sobre o metabolismo do ácido
retinóico nos lobos dorsais e laterais da próstata de ratos
adultos"
"Effects of chronic ethanol consumption on the activity
of MMP-2 /MMP-9 and on retinoic acid metabolism in
the dorsal and lateral prostate lobes of adult rats"
Resumo
Pesquisadores têm mostrado que o consumo crônico de etanol altera a concentração do ácido retinóico, metabólito ativo da vitamina A, em muitos órgãos, incluindo a próstata. O ácido retinóico é essencial para o desenvolvimento normal da próstata e para a manutenção de sua homeostase. Alterações na concentração e no metabolismo do ácido retinóico estão relacionadas com o desenvolvimento de lesão na próstata. Adicionalmente, a atividade de metaloproteinases da matriz extracelular (MMPs), também está relacionada com o desenvolvimento de alterações na próstata. Assim, o presente trabalho teve por objetivo descrever os efeitos dos consumos, baixo e alto, de etanol sobre as proteínas envolvidas na síntese e no catabolismo do ácido retinóico (artigo I), bem como, sobre a atividade enzimática das MMPs (artigo II) nos lobos dorsais e laterais da próstata.Vinte ratos adultos (~ 90 dias de idade) de cada variedade, UChA e UChB, foram divididos nos grupos (n=10/grupo): UChA (consumo baixo de etanol, 0,2-2 g/kg/dia), UChAC (ratos que não consumiram etanol); UChB (consumo alto de etanol, > 2g/kg/dia), UChBC (ratos que não consumiram etanol).Após o período experimental (~ 150 dias de idade), os ratos foram eutanasiados por decapitação e os lobos dorsais e laterais das próstatas foram coletados e dissecados: (1) para avaliar os níveis e a localização das proteínas ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1, CYP2E1, através de western blot e imuno-histoquímica, bem como, a atividade catabólica das CYP26A1, CYP26B1, CYP2E1 por ensaio bioquímico e quantificação por HPLC-MS/MS; (2) e para avaliar a atividade da MMP-2 e da MMP-9, e os níveis dos inibidores teciduais de metaloproteinases (TIMP-1/ TIMP-2), através de zimografia e Elisa, respectivamente. No grupo UChA, a ALDH1A3 aumentou na próstata dorsal, enquanto as proteínas ALDH1A1 e ALDH1A2 diminuíram na próstata
lateral. No grupo UChB, as proteínas ALDH1A1, ALDH1A2, e ALDH1A3 aumentaram na próstata dorsal, enquanto a ALDH1A3 diminuiu no lobo lateral. A concentração do ácido retinóico aumentou, indicando diminuição da atividade da CYP2E1, e diminuiu quando se avaliou a CYP26, indicando aumento de sua atividade na próstata dorsal do UChB. Além disso, o ácido retinóico diminuiu quando se avaliou a atividade de CYP total nos grupos experimentais, sendo somente aumentado na próstata lateral do UChA. O consumo baixo de etanol (grupo UChA) diminuiu a atividade das MMP-2 e MMP-9 e o nível das TIMP-2 e TIMP-1 na próstata lateral, enquanto que na próstata dorsal o etanol diminuiu a atividade de MMP-2 e o nível de TIMP-1. Por outro lado, no grupo UChB, o etanol diminuiu somente a atividade da MMP-9 na próstata lateral e não alterou os níveis de TIMP-1 e TIMP-2.Nossos resultados indicam que o etanol modula a síntese e o catabolismo do ácido retinóico na próstata do rato de modo dependente de sua concentração. Além disso, o consumo crônico e baixo de etanol diminui a atividade das metaloproteinases -2 e -9, sendo a próstata lateral o lobo afetado e, portanto, mais susceptível a estas alterações, do que o lobo prostático dorsal.
Abstract
Researchers have shown that chronic ethanol consumption alters the retinoic acid concentration, an active metabolite of vitamin A, in many organs including the prostate. The retinoic acid is essential for the normal development of prostate and for maintaining its glandular homeostasis. Changes in concentration and metabolism of retinoic acid are related to lesion development in the prostate. Additionally, the activity of matrix metalloproteinases (MMPs), also relates to development of alterations in prostate. Thus, this study aimed to describe the effects of low and high doses of ethanol consumption, on the proteins involved in the synthesis and catabolism of retinoic acid (Article I), as well as on the enzymatic activity of MMPs (Article II) the dorsal and lateral lobes of the prostate. Twenty adult rats (~ 90 days old) of each variety, UChA and UChB, were divided into groups (n = 10 / group): UChA (low ethanol consumption, 0.2-2 g /kg / day), UChAC (rats not consumed ethanol); UChB (high ethanol consumption, > 2 g/ kg/ day), UChBC (rats not consumed ethanol). After the experimental period (~ 150 days old), the rats were euthanized by decapitation and dorsal and lateral lobes of the prostates were collected and dissected: (1) for evaluate the levels and location of the proteins ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1, CYP2E1, by western blot and immunohistochemistry, as well as, catabolic activity of CYP26A1, CYP26B1, CYP2E1 by biochemical assay and quantification by HPLC–MS/MS; (2) and to evaluate the activity of MMP-2 and MMP-9, and the levels of tissue inhibitors of metalloproteinases (TIMP-1 / TIMP-2) using zymography and ELISA, respectively. In the UChA group, ALDH1A3 increased in dorsal prostate, while the proteins ALDH1A2 and ALDH1A1 decreased in the lateral prostate. In the UChB group, the proteins ALDH1A1, ALDH1A2 and ALDH1A3 increased in the
dorsal prostate, while ALDH1A3 decreased in the lateral lobe. The concentration of retinoic acid increased, indicating a decrease in the CYP2E1 activity, and decreased when evaluated CYP26, indicating increased of CYP26 activity in the UChB dorsal prostate. Furthermore, the retinoic acid decreased when assessing the CYP total activity in the experimental groups, but only increased in the lateral prostate of UChA. The low ethanol consumption (UChA group) reduced the activities of MMP-2 and MMP-9 and the levels of TIMP-2 and TIMP-1 in the lateral prostate, while dorsal prostate the ethanol decreased the MMP-2 activity and the level of TIMP-1. On the other hand, in the UChB group, ethanol only decreased the activity of MMP-9 in the lateral prostate and did not alter the levels of TIMP-1 and TIMP-2. Our results indicate that ethanol modulates the synthesis and catabolism of retinoic acid in the rat prostate in a concentration-dependent manner. In addition, the chronic and low consumption of ethanol decreases the activity of metalloproteinases -2 and -9 in the lateral lobe prostate, showing that this organ is more susceptible to these changes than dorsal lobe prostate.
Sumário
Resumo... vii Abstract...viii Introdução...1 Justificativas e Objetivos...6 Artigo I... 7 Artigo II ...35 Conclusão Geral...53 Referências ...54 Anexos...60“A tarefa não é tanto ver aquilo que ninguém viu, mas pensar
o que ninguém ainda pensou sobre aquilo que todo mundo vê”
Arthur Schopenhauer
“A persistência é o menor caminho do êxito”
Charles Chaplin
A Deus, pois através de sua imensa benevolência me
permitiu realizar esta tese. Seu amor, proteção e cuidado foram
e são essenciais para mim nesta jornada. Aos meus pais, às
minhas irmãs, ao meu marido, Francisco, e ao meu filho Pedro,
o qual Deus está gerando em meu ventre e que logo estará em
meus braços. Vocês são as maiores “obras” de Deus em minha
vida. Amo vocês!
“Sejam humildes debaixo da poderosa mão de Deus para que ele os
honre no tempo certo”
I Pedro 5:6
Agradecimentos
Aos meus orientadores, Prof. Dr. Francisco Eduardo Martinez e Prof. Dr. Sérgio Luis Felisbino, pela amizade, por terem acreditado neste trabalho e por todas as discussões e correções no sentido de melhorar este trabalho.
Aos Profs., Alexandre L. R. de Oliveira,Raquel Fantin Domeniconi e Taize Machado Augusto, por aceitarem participar do meu exame de qualificação e pelas considerações feitas para melhorar este trabalho.
Ao Programa de Pós-graduação em Biologia Celular e Estrutural da Unicamp e seus docentes, pela qualidade do programa e pelo excelente nível de profissionais.
À CAPES pelo importante apoio financeiro ao programa.
À FAPESP, pelo importante apoio financeiro concedido na forma de auxílio à pesquisa (2011/13713-0) e bolsa de doutorado no país (2011/03394-4).
Aos Profs., Sergio Pereira, Arielle Cristina Arena, Raquel Fantin Domeniconi e Bruno César Schimming, por aceitarem ser membros da banca de defesa desta tese.
Ao Prof. Dr. Gustavo de Almeida Chuffa, pela amizade e por me ajudar e redigir os artigos científicos.
À Profa. Dra. Patricia Fernanda Felipe Pinheiro e à Profa. Dra. Raquel Fantin Domeniconi, por todo apoio e incentivo durante o curso de doutorado, pelos esclarecimentos durante meu estágio na disciplina de anatomia humana e também pela amizade.
A todos os docentes do Departamento de Anatomia Humana, pela amizade e agradável convivência.
Aos amigos da pós-graduação, Leonardo e Fabiana, por todas as ajudas, mas principalmente pela amizade.
Aos funcionários do Departamento de Anatomia: Cristiane, Paulo, Luciano e Gelson por toda ajuda e pelos momentos de descontração.
Às minhas amigas, Paula, Dorcas e Carol, por sempre torcerem por mim e pela amizade de longa data.
A toda minha família (avós, tios e primos), por toda palavra de incentivo, pelo carinho e por fazerem parte da minha história.
À minha sogra e ao meu cunhado, pelo apoio e incentivo. Ao meu cunhado Dú, pelo apoio e momentos de descontração.
Enfim, a todas as pessoas que direta ou indiretamente me ajudaram durante este percurso, muito obrigada.
Introdução
O consumo crônico de álcool é um dos tipos mais prevalentes de dependência de droga no mundo (Rehm et al., 2006). A ingestão de álcool, por tempo prolongado, é importante fator de risco para o desencadeamento de diversos problemas de saúde, incluindo o desenvolvimento de câncer (Rehm, 2011). Os mecanismos propostos para a carcinogênese relacionada ao consumo crônico de etanol incluem: aumento da geração de radicais livres e acetaldeído, que tem efeito mutagênico e carcinogênico, indução do citocromo microssomal P4502E1 (CYP), ativação de enzimas que atuam em diferentes procarcinogênicos, o metabolismo anormal do retinol (vitamina A) e do seu metabólito ácido retinóico (Pöschl & Seitz, 2004; Wang, 2005; Seitz & Stickel, 2007).
Para explicar os efeitos do consumo de bebidas alcoólicas por humanos, pesquisadores têm utilizado modelos animais com preferência ao consumo de etanol (Cândido et al., 2007). As linhagens de ratos UCh (UCh = Universidade do Chile) foram selecionados, a partir de ratos Wistar, para ingerirem voluntariamente etanol e há décadas são mantidos sob cruzamento inbreeding (Mardones, 1983; Martinez et al., 2000). Os modelos UChA (consumo baixo de etanol) e UChB (consumo alto de etanol) possuem predisposição genética para ingerir voluntariamente etanol a 10% e são considerados excelentes modelos para explicar aspectos bioquímicos, fisiológicos e morfológicos relacionados ao consumo de álcool.
O consumo crônico de etanol provoca diversas alterações morfofisiológicas nos órgãos do sistema genital masculino (Van Thiel et al., 1979; Martinez et al., 2000; 2001a,b). Na próstata, as alterações provocadas pelo etanol são caracterizadas por atrofia
do epitélio, interrupção da homeostase estroma-epitélio, neoplasia intra-epitelial, indução de processos inflamatórios e aumento nos níveis do ácido retinóico (Martinez et al., 2000; 2001a; Cândido et al., 2007; Mendes et al., 2011; Fontanelli et al., 2013). Além disso, estudos epidemiológicos têm mostrado uma relação entre o consumo crônico e excessivo de álcool com o risco de desenvolvimento de câncer de próstata (Hirayama, 1992; Sharpe& Siemiatycki, 2001).
A próstata é uma glândula sexual acessória masculina frequentemente afetada pela hiperplasia benigna e pelo câncer (Guess, 2001). Nos roedores, é formada por três pares de lobos: ventral, lateral e dorsal (Prins, 1992). O lobo dorsal e o lateral, também denominados de lobo dorsolateral, apresentam analogia com a zona periférica da próstata humana, local de maior incidência de carcinomas, e por isso tem sido alvo de estudos por muitos pesquisadores (Roy-Burman et al., 2004). Microscopicamente, a próstata é constituída por células epiteliais e estroma. O epitélio da próstata é formado por três tipos celulares: basal, secretor (luminal) e neuroendócrino. O estroma prostático é formado por um complexo arranjo de células do estroma e componentes da matriz extracelular (MEC), associados a fatores de crescimento, moléculas reguladoras e enzimas remodeladoras. As células do estroma e a matriz extracelular constituem um microambiente que controla o crescimento e a diferenciação funcional de células adjacentes e assim, contribuem para a manutenção da forma e função do tecido (Narbaitz, 1975; Labat-Robert et al., 1990; Tuxhorn et al., 2001). Além disso, a degradação da MEC é um passo importante para o crescimento, invasão e metástase da célula cancerosa e, envolve um conjunto especial de enzimas denominadas de metaloproteinases de matriz (MMP), endopeptidases metal-dependente, capazes de degradar colágeno, elastina, lamininas, fibronectina e proteoglicanos (Matrisian, 1990; Nagle, 2004).
O retinol (vitamina A) e seu metabólito ativo, ácido retinóico, são importantes para a manutenção das funções biológicas dos vertebrados incluindo desenvolvimento, diferenciação e reprodução (Fields et al., 2007). No sistema genital, o retinol e o ácido retinóico são essenciais para a manutenção do tecido epitelial da glândula seminal e do testículo e são indispensáveis para a espermatogênese (Huang et al., 1983; Ghyselinck et al., 1999). Na próstata, são importantes para as fases de desenvolvimento, diferenciação das células epiteliais, bem como, para a manutenção de sua homeostase (Lasnitzki & Goodman, 1974; Vezina et al., 2008).
O etanol interfere no metabolismo do ácido retinóico e altera sua concentração em diversos tecidos (Lieber, 1991;Wang, 1999). As alterações provocadas pelo etanol na concentração do ácido retinóico diferem nos tecidos. No fígado, a exposição ao etanol diminui os níveis de ésteres de retinol (a forma de estocagem do retinol), retinol e de ácido retinóico (Chung, 2001). Por outro lado, no hipocampo, no testículo e no soro, a concentração do ácido retinóico aumenta frente à exposição crônica ao etanol (Kane et al., 2009). Recentemente, nós descrevemos aumento da concentração do ácido retinóico no lobo dorsolateral da próstata de ratos que apresentam consumo alto e crônico de etanol (Fontanelli et al., 2013). No entanto, os mecanismos pelo qual o etanol altera a concentração do ácido retinóico na próstata não são conhecidos.
O etanol induz alterações moleculares que somadas à perda da homeostase do ácido retinóico podem contribuir para o desenvolvimento de patologias como, por exemplo, o câncer e a hiperplasia prostática benigna (HPB) (Wang, 2005; Seitz & Stickel, 2007). Uma relação parece existir entre o consumo crônico de etanol e o câncer de próstata (CaP), no entanto, os mecanismos pelo qual o etanol pode contribuir para o desenvolvimento do câncer não são conhecidos. Alterações na concentração e na via metabólica do ácido
retinóico foram observadas durante a HPB e o CaP, indicando que esta via possa ser importante no desenvolvimento de patologias prostática (Pasquali et al., 1996; Richter et al., 2002).
Adicionalmente, pesquisas recentes têm mostrado que o ácido retinóico atua sobre a expressão e a atividade das MMPs, podendo aumentar a degradação da MEC e o potencial de migração celular e ainda participar na progressão do desenvolvimento do câncer (Dutta et al., 2009; Lackey & Hoag, 2010).É sabido que o aumento da atividade das MMPs bem como o aumento dos níveis das MMP-2 e MMP-9 são associados a mau prognóstico e ao avanço do CaP, respectivamente, e podem funcionar como marcadores da progressão da doença (Sauer et al., 2004; Semaan et al., 2005).
A via metabólica dos retinóides (Figura 1) consiste em gerar moléculas com potencial função aos tecidos e a degradar seu excesso (Kumar et al., 2011). A homeostase do ácido retinóico depende de proteínas relacionadas à sua síntese e degradação (Kumar et al., 2011). No ambiente intracelular, o retinol pode ser oxidado ou estocado sob a forma de ésteres de retinol em gotas de gordura (Miano& Berch, 2000). A oxidação reversível do retinol pela ação da enzima álcool/retinol desidrogenase (ADH/RDH), gera primeiramente retinal e, posteriormente, a oxidação irreversível do retinal pela aldeído/retinaldeído desidrogenase (ALDH/RALDH), gera o ácido retinóico (Molotkov & Duester, 2002). O ácido retinóico regula a proliferação celular, a diferenciação e a apoptose, ligando-se a receptores nucleares, os quais são membros da superfamília de receptores de hormônios esteróides. A ligação do ácido retinóico com seus receptores faz com que eles atuem como fatores de transcrição, modulando a expressão gênica (Wang, 2005; Pöschl & Seitz, 2004).
Dentro da família da enzima ALDH, responsável pela oxidação irreversível do retinal a ácido retinóico, a ALDH1 é apontada como eficiente na geração do ácido retinóico (Lee et al., 1991) e possui três isoformas: ALDH1A1, ALDH1A2 e ALDH1A3 (Vasiliou et al., 2004). As enzimas da família 26 do complexo citocromo P450 realizam o catabolismo do ácido retinóico nos tecidos e assim, controlam sua concentração (Noy, 2000). As enzimas CYP26A1 e CYP26B1 participam do catabolismo do ácido retinóico na próstata e se apresentam expressas desde o desenvolvimento prostático (Vezina et al., 2008). Adicionalmente, tem sido mostrado que a enzima CYP2E1, além de atuar no metabolismo do etanol, também participa do metabolismo do ácido retinóico (Liu et al., 2001; Chung et al., 2009).
Figura 1.Via metabólicados retinóides.O all-trans-retinol (a forma de álcoolda vitaminaA)pode ser convertido emésteres de
retinolpara ser armazenadoatravés da açãoda lecitina-retinol aciltransferase (LRAT) e da proteina que se liga ao retinol celular tipo-1 (CRBP1). Alternativamente,all-trans-retinol pode seroxidado aall-trans-retinaldeídopor umaálcool desidrogenase(ADH) ou retinol
desidrogenase (RDH) utilizando NADcomo cofator. Oall-transretinaldeídotambém pode ser geradopor clivagemdobeta-caroteno (provitaminaA)pela ação daenzima beta-caroteno 15,15'-monooxigenase (CMO1). A all-trans-retinaldeídopode ser reduzida
atrans-retinol através da ação deRDHsque preferencialmenteutilizamNADPHcomo cofatorou poraldo-keto redutases(AKR). A all-transretinaldeído pode ser aindaoxidada aall-trans-ácido retinóico (AR) por retinaldeído desidrogenases(RALDH1, RALDH2e
RALDH3) que são membrosda famíliaaldeído desidrogenase(ALDH1A1, ALDH1A2eALDH1A3, respectivamente). ARpode ser aindaoxidado a4-hidroxi-ácido retinóico pelo citocromoP450 (CYP26A1, CYP26B1, e CYP26C1), que é considerado o primeiro passoda degradaçãoAR, pois leva a metabólitosmais facilmenteexcretado.ARpodeiniciar um eventode sinalizaçãoatravés da ligação
Justificativa
Estudos epidemiológicos e experimentais demonstram uma relação entre o consumo crônico de etanol com o desenvolvimento de lesões na próstata. O consumo crônico de etanol aumenta a concentração do ácido retinóico no tecido prostático. O ácido retinóico é essencial para a manutenção da homeostase da próstata. A interferência do etanol no metabolismo do ácido retinóico é descrito ser um mecanismo importante para o desenvolvimento de patologias em alguns tecidos. Adicionalmente, pesquisas demonstram que o metabolismo e a concentração do ácido retinóico são alterados durante a hiperplasia prostática benigna e o câncer de próstata, e sugerem que esta via seja importante para o desenvolvimento destas lesões. Tendo em vista que o consumo crônico de etanol é um dos mais graves problemas de saúde mundial, espera-se poder contribuir na elucidação das alterações provocadas pelo etanol sobre a via metabólica do ácido retinóico e sobre a atividade de enzimas remodeladoras da MEC do estroma prostático, objetivando colaborar com as pesquisas que relacionam o etanol com as lesões da próstata, além de trazer novos esclarecimentos sobre as disfunções induzidas pelo etanol nessa glândula.
Objetivos
O presente trabalho teve como objetivos:
1. Analisar os efeitos do consumo de etanol sobre a síntese e o catabolismo do ácido retinóico nos lobos dorsais e laterais da próstata de ratos consumidores de etanol; 2. Avaliar os efeitos do etanol sobre a atividade enzimática das MMP-2 e MMP-9 nos
Artigo I
Ethanol intake modulates the synthesis and catabolism of
retinoic acid in the prostate tissue
Ethanol intake modulates the synthesis and catabolism of retinoic acid in the prostate tissue
Beatriz Aparecida Fioruci-Fontanelli1,2; Luiz Gustavo A. Chuffa1; Leonardo O. Mendes1,2; Patricia Fernanda F. Pinheiro1; Luis Antônio Justulin Jr3; Sérgio Luis Felisbino3 and Francisco Eduardo Martinez1*
1
Department of Anatomy, Institute of Biosciences, UNESP – Univ Estadual Paulista, Botucatu, SP, Brazil;
2
Structural and Cell Biology Program, UNICAMP, Campinas, SP, Brazil;
3
Department of Morphology, Institute of Biosciences, UNESP – Univ Estadual Paulista, Botucatu, SP, Brazil;
*Corresponding author:
Francisco Eduardo Martinez, Department of Anatomy, Institute of Bioscience, UNESP – Univ Estadual Paulista, P.O. Box 510, Postal Code: 18618-970, Rubião Júnior, s/n, Botucatu, SP – Brazil, Telephone number: +55 (14) 3880-0024, Fax: +55 (14) 3811-6361,
Abstract
All-trans-retinoic acid (atRA) maintains physiological stability of the prostate, and
we reported in a study previously conducted that ethanol intake increases atRA in the rat prostate; however the mechanisms underlying these changes are unknown. We evaluated the impact of a low- and high-dose ethanol intake (UChA and UChB strain) on the metabolism of atRA in the dorsal and lateral prostate. ALDH1A3 increased in the dorsal prostate of UChA animals while ALDH1A1 and ALDH1A2 decreased in the lateral prostate. In UChB animals, ALDH1A1, ALDH1A2, and ALDH1A3 increased in the dorsal prostate, and ALDH1A3 decreased in the lateral prostate. The concentration of atRA increased with the low activity of CYP2E1 and decreased with high CYP26 activity in the UChB dorsal prostate. Conversely, atRA was found to decrease when the activity of CYP total in the UChA lateral prostate was increased. Ethanol modulates the synthesis and catabolism of atRA in the rat prostate in a concentration-dependent manner.
1. Introduction
Retinoic acid (RA), an active metabolite of retinol (vitamin A), is responsible for the maintenance of several biological functions in vertebrates, including reproduction [7]. RA acts through four distinct isomers: all-trans-RA (atRA), 9-cis-RA, 13-cis-RA, and 9,13-dicis-RA. Among these, atRA is the most abundant and it is considered to be the biologically active isomer [2].
In the prostate, atRA controls development of prostatic buds, differentiation of epithelial cells, and contributes to the maintenance of homeostasis in the adult prostate [13, 32]. In conjunction with members of the steroid hormone receptor superfamily, atRA acts as a transcription factor, consequently regulating cell differentiation, apoptosis, and proliferation [21].
The concentration of atRA is regulated by enzymes responsible for its synthesis and catabolism [12]. The synthesis of atRA occurs by the irreversible oxidation of retinal, a derivative of retinol oxidation, by aldehyde dehydrogenase (ALDH), also known as retinaldehyde dehydrogenase [19]. The proteins of ALDH1A family (ALDH1A1, ALDH1A2, and ALDH1A3) are responsible for RA synthesis [14, 31]. On the other hand, cytochrome P450 enzyme, family 26, breaks down RA in the tissues and reduces its levels [19]. The CYP26 family comprises three isoforms: CYP26A1, CYP26B1, and CYP26C1 [26]. CYP26A1 and CYP26B1 enzymes metabolize the atRA isomer [3], whereas CYP26C1 appears to prefer 9-cis-RA as a substrate [29]. Additionally, CYP2E1 enzyme is involved mainly in the catabolism of ethanol, but also participates in the excretion of RA upon induction by ethanol [6, 16].
Ethanol has been shown to interfere with RA metabolism and change its concentration in tissues differentially [15, 33]. In the liver, ethanol decreases the atRA
levels [5], while increasing it in the hippocampus, testis, and serum [10]. In the prostate, only a high dose of chronic ethanol ingestion increases atRA, while a lower dose of ethanol does not [8]. Importantly, changes in the atRA pathway and atRA levels may contribute to the development of benign prostatic hyperplasia (BPH) and prostate cancer [20, 24]. Therefore, the knowledge of how ethanol modulates the atRA metabolism in the prostate is important and can yield new insights into damage caused by ethanol in this organ.
Our aim was to evaluate the effects of both high and low doses of ethanol intake upon the proteins responsible for synthesis and catabolism of atRA in the dorsal and lateral lobes of the UCh rat prostate.
2. Materials and Methods
2.1 Animals, groups, and diet
Forty UCh (University of Chile) male rats, weighing between 280–330 g (~90 days old), were obtained from the Department of Anatomy, Bioscience Institute, Campus of Botucatu, UNESP- Universidade Estadual Paulista (IBB/UNESP). The UCh strain is an ethanol-preferring rat displaying voluntary ethanol intake [17]. There are two UCh rat strains, UChA and UChB, which exhibit low ethanol consumption (0.2-2 g/kg/day) and high ethanol consumption (4-8 g/kg/day) of 10% (v/v) ethanol solution, respectively [22]. Twenty adults of each variety, UChA and UChB, weighing between 280-330 g (~90 days old), were obtained from the Department of Anatomy, Bioscience Institute, Campus of Botucatu, UNESP – Univ Estadual Paulista (IBB/UNESP). In this study, the animals were divided into four sub-groups (n=10/group): UChA: low-ethanol intake, UChAC: no ethanol intake, UChB: high-ethanol intake, UChBC: no ethanol intake.
The ethanol ingestion was measured weekly throughout the experimental period (60 days) using a marked test tube. All rats were housed in individual cages in a temperature- and humidity-controlled room under a 12-h light-dark cycle, had free access to filtered tap water, and were fed with standard rodent chow Nuvital® (Nuvilab CR-1). At 120 days old, the animals were euthanized in CO2 chamber. The experimental protocol was approved by the Ethical Committee of the Institute of Bioscience, Campus of Botucatu, SP, Brazil (IBB/UNESP, Protocol nº 340/2011).
2.2 Protein extraction and western blot analysis
After 60 days of ethanol consumption, the dorsal and lateral lobes of the prostate of UChA and UChB rats were collected and immediately frozen in liquid nitrogen and stored at -80°C. Extraction of proteins and western blot were performed using methods described previously [4]. Antibodies against ALDH1A1 (1:2000), ALDH1A2 (1:250), and ALDH1A3 (1:250) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-CYP2E1 (1:250), anti-CYP26A1 (1:500), and anti-CYP26B1 (1:500) were purchased from Abcam (Inc., Cambridge, MA, USA). After incubation with primary antibody, the membranes were incubated for 2 h at RT with rabbit or mouse HRP-conjugated secondary antibodies (diluted 1:1000 in 1% BSA; Sigma, St. Louis, MO, USA). Blots were revealed using ECL Western Blotting system (Amersham) and analyzed with a densitometer (G-BOX calibrated imaging densitometer, Syngen). β-Actin was used as an endogenous control and all results are expressed as mean ± SEM. Immunoblotting concentrations (%) were represented as optical densitometry values (band intensity/β-actin ratio).
2.3 Immunohistochemistry
Dorsal and lateral prostate tissue sections (4 µm thick), paraffin embedded, of the 5 samples/group were deparaffinized and treated in a water bath for 30 min, at 95°C, in Tris/EDTA + Tween 20 (pH 9.0) for antigen recovery. After cooling, the sections were incubated with monoclonal antibodies: anti-ALDH1A1 (1:100), anti-CYP26A1 (1:100), anti-CYP26B1 (1:100), and anti-CYP2E1 (1:70) (Abcam, Inc., Cambridge, MA, USA), at 4 °C overnight, subsequently rinsed inPBS and incubated with anti-rabbit HRP-conjugated secondary antibody (Sigma, St. Louis, MO, USA) for 1 h. The color reaction was developed with 3,3΄-diaminobenzidine tetrahydrochloride (DAB) (Sigma Chemical Co.). The reaction was stopped in distilled water, and sections were counterstained with hematoxylin. The sections were analyzed and photographed using an Olympus (BX-41) microscope, equipped with a DP-12 digital camera (Olympus, Inc., Japan).
2.4 Immunofluorescence
Frozen sections of dorsal and lateral lobes of rat prostate were mounted on precooled chucks (-20 °C) in a Leica cryostat, and 8 μm sections were thaw-mounted on silane-coated glass slides. For fixing and permeabilizing the tissue, the sections were placed in an ice-cold acetone/methanol solution (2:1, v/v), for 10 min. After the sections were washed with TBS buffer (pH 7.4) 3 times (5 min per wash), and blocked using 5% non-fat milk for 1 h. The sections were incubated with monoclonal antibodies anti-ALDH1A2 and anti-ALDH1A3 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and were placed in a moist chamber at 4 °C overnight. The fluorescein isothiocyanate (FITC)-labeled anti-goat secondary antibody (Sigma, St.Louis, MO, USA) was used against the ALDH1A2 and ALDH1A3 antibodies. The slides were mounted with Vectashield
containing 4’,6-diamido-2-phenylindole hydrochloride (Vector Laboratories, Inc.), to visualize nuclei. Photographs of slides were taken using a laser scanning confocal microscope (Leica, Inc., Wetzlar, Germany).
2.5 Subcellular fractions from prostate tissue
Initially, dorsal and lateral prostates were homogenized in 500 µl of buffer A (Tris-HCl (10 mM), sacarose 10% (w/w), EDTA (1mM), DTT (1.5 mM), pH 7.4), using a tissue homogenizer (Ika, T 10 basic Ultra-Turrax, Staufen, Germany). The homogenates were centrifuged (4 °C, 14,000 rpm, 20 min.), and the supernatants were pipetted, and subjected to differential centrifugation to isolate the microsomal fraction, according to the methoddescribed by Kane et al. [10]. After differential centrifugation (4 °C, 100,000 g, 1 h), supernatants were removed (cytosol) and the pellet (microsomes) was resuspended and hand-homogenized in buffer A. The total protein concentration was determined using the Bradford assay.
2.6 Microsomal activity
Assessment of activity of P450 enzymes of the microsomal fraction (CYP26A1, CYP26B1 and CYP2E1), was performed in triplicate with or without CYP enzyme inhibitors. Initially, the buffer (50 mM Tris-HCl, 150 mMKCl, 5 mM MgCl2, pH 7.4), with
NADPH included, was placed in glass tubes at a total volume of 0.5 ml and agitated at 65 rpm in a water bath at 37 °C for 3 min. The reaction was started by adding 5 μl of substrate (2 nmol of atRA dissolved in DMSO) in samples containing prostatic microsomes (200 μg protein) and incubated at 37 °C in a shaking water bath (65 rpm) for 15 min. Before incubation with the substrate (atRA), to evaluate the effects of ethanol consumption on the
catabolism of the CYP (CYP26A1, CYP26B1 and CYP2E1), the samples were preincubated for 10 min with specific inhibitors of CYP, with some adjustments to the method described in Liu et al. [16]. The specific inhibitors of CYP used were chlormethiazole (specific inhibitor of CYP2E1), α-naphthoflavone (of CYP1A2), troleandomycin (of the CYP3A family), and liarozole (of the CYP26 family), followed by incubation with atRA for 15 min. [16].
After incubation for 15 minutes, atRA was extracted from the samples, quantified by LC-tandem mass spectrometry (MS/MS) and analyzed in the Laboratory for analysis of retinoids at the Department of Nutritional Science and Toxicology, University of California-Berkeley, USA.
2.7 Statistical analysis
For the UChA and UChAC groups, the proteins were evaluated using an unpaired t test, and for the UChB and UChBC groups, proteins were compared using the Mann-Whitney test. Differences were considered significant at P< 0.05. The statistical software used was Graph Pad InStat version 3 (Graph Pad Software, San Diego, CA, USA), and Sigma Plot version 11 (Systat Software, Canada, USA) was used for graphic design.
3. Results
3.1 Levels and localization of proteins involved in atRA metabolism
The proteins were quantified in the dorsal and lateral prostates of all experimental groups. The proteins ALDH1A1, ALDH1A2, CYP2E1, CYP26A1, and CYP26B1 did not change in the dorsal prostate of UChA compared to UChAC, while ALDH1A3 increased with ethanol consumption (UChA) (Fig. 1). On the other hand, in the lateral prostate the
ALDH1A1, ALDH1A2, and CYP2E1 decreased (Fig. 2) in the UChA group compared to its control group, while ALDH1A3, CYP26A1, and CYP26B1 were unaltered (Fig. 2).
In the dorsal prostate of UChB group, ALDH1A1, ALDH1A2, and ALDH1A3 increased, while CYP2E1, CYP26A1, and CYP26B1 did not change compared to UChBC group (Fig. 3, 4). In the lateral prostate, ALDH1A3 decreased with other proteins remaining unchanged in the UChB compared to UChBC (Fig. 3, 5). The localization of the proteins is shown in the Tables 1 and 2, as well as Figures 3 and 6.
3.2 Evaluation CYP activity through of changes in atRA rate
The activity of CyP is not altered in the dorsal prostate of UChA compared to its control group (UChAC), since the concentration of the atRA is unchanged (Fig. 7 a). In the lateral prostate, the activity of CYP26 family did not change the atRA concentration. On the other hand, there was decreased CYP2E1 activity resulting in increase of atRA levels in the UChA compared to UChAC (Fig. 7 b). The activity of other CYP forms (nonspecific) increased, leading to a lower atRA concentration (Fig. 7 b) in the lateral prostate of UChA compared to UChAC.
In the dorsal prostate of UChB, the CYP26 activity increased compared to UChBC, resulting in low atRA levels (Fig. 8 a). However, the CYP2E1 activity decreased after ethanol consumption (UChB) compared to control group (UChBC), causing an increase in
atRA (Fig. 8 a). In the lateral prostate, no change was observed in CYP activity and
4. Discussion
The ethanol-induced changes in the levels of the ALDH1A family proteins in the dorsal and lateral lobes of the prostate. The isoforms of ALDH1A (ALDH1A1, ALDH1A2 and ALDH1A3) use retinal as a substrate to produce retinoic acid [19]. So, when they are altered, it gives rise to differences in the concentration of retinoic acid in different tissues.
The high ethanol consumption showed increase the retinoic acid concentration in the prostate in study realized previously [8]. The findings of this study shows us the mechanism by which ethanol alters the levels of retinoic acid in the prostate and thus can contribute with research linking prostatic lesions and changes of retinoic acid metabolism, in addition to bringing new insights on damage caused by ethanol consumption in the prostate. The increase of ALDH1A1, ALDH1A2, and ALDH1A3 in the UChB dorsal prostate may explain the increase in the concentration of retinoic acid in the prostate. In the lateral prostate, the high ethanol consumption decreased ALDH1A3, but did not alter either ALDH1A1 or ALDH1A2, and this may not have been sufficient to cause an increase in levels of atRA. Thus, high ethanol consumption affects atRA synthesis more in the dorsal prostate than in the lateral prostate. On the other hand, a previous study, showed that a low ethanol consumption did not alter the concentration of retinoic acid in the prostate [8]. Additionally, ALDH1A1 and ALDH1A2 are not altered in the dorsal prostate with low ethanol consumption and the sole increase in ALDH1A3 is insufficient to alter the retinoic acid concentration.
The imbalance in retinoic acid metabolism is closely related to development of prostate diseases [9, 11, 20, 23]. Frequently, retinoic acid levels and proteins of family ALDH1A are shown to be altered in prostate cancer and BPH, and are thus used as indicators of prostatic lesions [11, 30].
ALDH1A isoforms are expressed in both human and rodent prostates [27, 28, 30]. However, the lateral and dorsal lobes of the mice prostates express different amounts of proteins ALDH1A1, ALDH1A2, and ALDH1A3 [30]. ALDH1A2, in the UChA lateral prostate, is expressed in the cytoplasm of epithelial and stromal cells and is absent in the nucleus. Similarly, there is a loss of the nuclear signal in case of prostate cancer [30]. In addition, ALDH1A2 was higher in the stroma than in the epithelium of dorsal and lateral lobes of the UChB prostates. ALDH1A3 increased in the UChA dorsal prostate, and the immunohistochemical analyses showed that it was due ALDH1A3 also being expressed in the cytoplasm of stromal cells. In the UChB, ALDH1A3 was much higherin dorsal prostate than that in UChBC, but did not have its localization altered. Researchers have shown that ethanol increases ALDH1A3 expression, suggesting that retinoic acid also increases consequently [10]. In the lateral prostate of UChB rats, ALDH1A3 decreased, and this can be explained by loss of cytoplasmic signal in the stromal cells. These results confirm that ethanol acts differentially on the retinoic acid pathway in different tissues [10].
The retinoic acid concentration in tissues may also be changed via cytochrome P450 proteins that are enzymes responsible for retinoic acid breakdown, generating less-active polar metabolites [18, 25]. Hepatic CYP2E1, in the presence of ethanol, increases the degradation of retinoic acid, reducing its concentration [16].This probably occurs as a compensatory mechanism to increase retinoic acid generated in the liver after prolonged exposure to ethanol [10].
CYP26A1 and CYP26B1 are essential for retinoic acid catabolism [1].The CYP26 activity increased in the dorsal prostate of UChB rats, causing decrease in retinoic acid. However, in the lateral and dorsal prostates of UChB group, CYP2E1 activity decreased, generating an increase in atRA. Additionally, the CYP total activity (nonspecific) also
decreased in the UChB dorsal prostate, increasing retinoic acid concentration. This decrease in the activities of CYP2E1 and CYP total (nonspecific) can contribute to an increase in atRA levels, mainly in dorsal prostate of UChB where an increase in ALDH1A proteins occurs. These results suggest that the reduced activity of these CYPs contribute to the increase of atRA, as described previously [8]. Moreover, the reduced activity of total CYP (nonspecific) suggests that other proteins of cytochrome P450 contribute to the increase of retinoic acid levels in the prostate.
We conclude that both low and high ethanol consumption alter the levels of proteins involved in the synthesis and catabolism of atRA in the prostate. Furthermore, high ethanol consumption has a greater impact on the proteins of the atRA metabolic pathway than low ethanol consumption, and thus it can alter the retinoic acid concentration in the prostate more significantly.
5. Acknowledgments
This work was supported by a grant from de Research Foundation of Sao Paulo State (2011/13713-0; 2011/03394-4). We thank Professor Joseph L. Napoli and Professor Charles Krois, Ph.D., of University of California, Berkeley, for analyzing the concentration of atRA.
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Table 1. Summary of the cellular distribution of the proteins in the UChA and UChAC groups
Dorsal Prostate Lateral Prostate
Protein UChAC UChA UChAC UChA
ALDH1A1 Nucleus and cytoplasm of epithelial cells
Nucleus and cytoplasm of ephitelial cells
Apical cytoplasm of ephitelial cells
Apical cytoplasm of epithelial cells ALDH1A2 Nucleus and cytoplasm of
epithelial cells and cytoplasm of stromal cells
Nucleus and cytoplasm of epithelial cells and cytoplasm of stromal cells
Nucleus and cytoplasm of epithelial cells and cytoplasm of stromal cells
Cytoplasm of epithelial and stromal cells
ALDH1A3 Nucleus and cytoplasm of epithelial cells
Nucleus and cytoplasm of epithelial cells and cytoplasm of stromal cells
Cytoplasm of epithelial and stromal cells
Cytoplasm of epithelial and stromal cells
CYP26A1 Nucleus and cytoplasm of epithelial cells
Nucleus and cytoplasm of epithelial cells
Cytoplasm of epithelial cells Nucleus and cytoplasm of epithelial cells
CYP26B1 Cytoplasm of epithelial cells Cytoplasm of epithelial cells Cytoplasm of epithelial cells Cytoplasm of epithelial cells CYP2E1 Cytoplasm and nucleus of
epithelial and stromal cells and blood vessels
Cytoplasm and nucleus of epithelial and stromal cells
and blood vessels
Cytoplasm and nucleus of epithelial and stromal cells
and blood vessels
Cytoplasm and nucleus of epithelial and stromal cells
Table 2. Summary of the cellular distribution of the proteins in the UChB and UChBC groups
Dorsal Prostate Lateral Prostate
Protein UChBC UChB UChBC UChB
ALDH1A1 Nucleus and cytoplasm of epithelial cells
Nucleus and cytoplasm of epithelial cells
Cytoplasm of stromal cell Cytoplasm of stromal cell ALDH1A2 Cytoplasm of epithelial and
stromal cells
Cytoplasm of epithelial and stromal cells
Cytoplasm of epithelial and stromal cells
Cytoplasm of epithelial and stromal cells ALDH1A3 Nucleus and cytoplasm of
epithelial cells
Nucleus and cytoplasm of epithelial cells
Cytoplasm of epithelial and stromal cells
Cytoplasm of epithelial cells
CYP26A1 Nucleus and cytoplasm of epithelial cells
Nucleus and cytoplasm of epithelial cells
Cytoplasm of epithelial cells and little nuclear
staining
Cytoplasm of epithelial cells and little nuclear
staining CYP26B1
Cytoplasm of epithelial and stromal cells and
blood vessels
Cytoplasm of epithelial and stromal cells and
blood vessels
Cytoplasm of epithelial and stromal cells and
blood vessels
Cytoplasm of epithelial and stromal cells and
blood vessels CYP2E1
Cytoplasm and nucleus of epithelial and stromal cells
and blood vessels
Cytoplasm and nucleus of epithelial and stromal cells
and blood vessels
Cytoplasm and nucleus of epithelial and stromal cells
and blood vessels
Cytoplasm and nucleus of epithelial and stromal cells
Figure Legends
Figure 1 Immunoblots and graphs of the proteins in the dorsal prostate of UChAC and
UChA rats (western blot). (A) ALDH1A1, ALDH1A2, and ALDH1A3; (B) CYP26A1, CYP26B1, and CYP2E1.(*P< 0.05)
Figure 2 Immunoblots and graphs of the proteins in the lateral prostate of UChAC and
UChA rats (western blot). (A) ALDH1A1, ALDH1A2, and ALDH1A3; (B) CYP26A1, CYP26B1, and CYP2E1.(*P< 0.05)
Figure 3 Localization of the atRA metabolism-related proteins in the dorsal and lateral
prostates of UChAC and UChA rats.Immunohistochemistry (A-D, M-Y) andImmunofluorescence (E-L). 40 X magnification, bars = 50 µm
Figure 4 Immunoblots and graphs of the proteins in the dorsal prostate of UChBC and
UChB rats (western blot). (A) ALDH1A1, ALDH1A2, and ALDH1A3; (B) CYP26A1, CYP26B1, and CYP2E1.(*P< 0.05)
Figure 5 Immunoblots and graphs of the proteins in the lateral prostate of UChBC and
UChB rats (western blot). (A) ALDH1A1, ALDH1A2, and ALDH1A3; (B) CYP26A1, CYP26B1, and CYP2E1 (*P< 0.05)
Figure 6 Localization of the atRA metabolism-related proteins in the dorsal and lateral
prostates of UChBC and UChB rats.Immunohistochemistry (A-D, M-Y) e Immunofluorescence (E-L). 40 X magnification, bars = 50 µm
Figure 7 Concentration of the atRAafter the catabolic activity of CYP26, CYP2E1 and
CYP total. (A)Dorsal prostate of the UChAC and UChAgroups; (B)Lateral prostate of the UChAC and UChAgroups. (**P< 0.01; *P< 0.05).
Figure 8 Concentration of the atRA after the catabolic activity of CYP26, CYP2E1 and
CYP total. (A)Dorsal prostate of the UChBC and UChBgroups; (B)Lateral prostate of the UChBC and UChBgroups.(* P< 0.05)
Artigo II
Tissue matrix metalloproteinases and tissue inhibitors of
metalloproteinases are altered in the rat prostate after
chronic alcohol intake
Submetido para publicação na revista AlcoholTissue matrix metalloproteinases and tissue inhibitors of metalloproteinases are altered in the rat prostate after chronic alcohol intake
Beatriz AparecidaFioruci-Fontanelli1,2; Luiz Gustavo A. Chuffa1; Leonardo O. Mendes1,2; Patricia Fernanda F. Pinheiro1; Flávia Karina Delella3; Cilmery S. Kurokawa4; Sérgio Luis Felisbino3and Francisco Eduardo Martinez1*.
1
Department of Anatomy, Institute of Biosciences, UNESP – Univ Estadual Paulista, Botucatu, SP, Brazil;
2
Structural and Cell Biology Program, UNICAMP, Campinas, SP,Brazil;
3
Department ofMorphology, Institute of Biosciences, UNESP – Univ Estadual Paulista, Botucatu, SP, Brazil;
4
Department of Pediatrics, Faculty of Medicine, UNESP – Univ Estadual Paulista, Botucatu, SP, Brazil.
*Corresponding author:
Francisco Eduardo Martinez, Department of Anatomy, Institute of Bioscience, UNESP – Univ Estadual Paulista, P.O. Box 510, Postal Code: 18618-970, Rubião Júnior, s/n, Botucatu, SP – Brazil, Telephone number: +55 (14) 3880-0024, Fax: +55 (14) 3811-6361,
Abstract
MMP-2 and MMP-9 (gelatinase A and B, respectively) are known to play key roles in tissue remodeling and repair through the degradation of many matrix proteins. In addition, the activities of MMP-9 and MMP-2 are precisely regulated by their tissue inhibitors, TIMP-1 and TIMP-2, respectively. The imbalance of MMPs/TIMPs contributes to the development of diseases such as arthritis, cancer cell invasion, metastasis and fibrosis. Ethanol alters MMP-2 and MMP-9 activities, which are responsible for the development of hepatic fibrosis. Although ethanol ingestion changes the MMP activity and TIMP levels, leading to pathologic processes, these effects in the prostate have not been explored. We investigated whether chronic ethanol ingestion, both low and high levels, is capable of altering the MMP-2 and MMP-9 activities and TIMP-2 and TIMP-1 levels in the dorsal and lateral prostate of UCh rats. Low chronic ethanol ingestion decreased MMP-2/MMP-9 activities and TIMP-2/TIMP-1 levels in the lateral prostate, whereas in the dorsal prostate, ingestion only decreased the activity of MMP-2 and the levels of TIMP-1. On the other hand, high chronic ethanol ingestion decreased only the activity of MMP-9 in the lateral prostate but did change the TIMP-1/TIMP-2 levels. Low chronic ethanol ingestion was more harmful to the prostate than high chronic ethanol ingestion and decreased the MMP-2 and MMP-9 activities in the lateral prostate, which was more susceptible. In addition, TIMP-1 and TIMP-2 positively regulated the MMPs in the lateral prostate, which, however, may be related to other events and not necessarily involved in the control of the MMPs.
Introduction
Chronic ethanol consumption has shown to cause several alterations in the reproductive organs of rats (Martinez et al., 2000; Chuffa et al., 2011; Chuffa et al., 2013). In the prostate, some damages caused by ethanol are characterized by atrophic epithelial cells (Martinez et al., 2001), a disturbance of normal stromal-epithelial homeostasis (Cândido et al., 2007), inflammation (Mendes et al., 2011), and increases in the concentration of retinoic acid, which controls events such as proliferation and apoptosis (Fontanelli et al., 2013).
The prostate gland produces and secretes several proteinases (Shimokawa et al., 2002), such as collagenase-like peptidase (Lukac and Koren, 1979) and gelatinolytic proteinases (Yin et al., 1990; Wilson et al., 1993), such as matrix metalloproteinase (MMP), that are important for turnover of the extracellular matrix components (ECM) as collagens, elastins, gelatin, matrix glycoproteins, and proteoglycan (Nagase, 1996). MMP-2 and MMP-9 (gelatinase A and B, respectively) are known to play key roles in tissue remodeling and repair through the degradation of many matrix proteins. The activity of MMP-2 and MMP-9 is regulated, respectively, by tissue inhibitors of metalloproteinases called of TIMP-2 and TIMP-1 (Gomes et al., 1997).
The imbalance of MMPs and TIMPs is an important stromal alteration involvedin development of diseases such as arthritis, cancer cell invasion, metastasis and fibrosis (Woessner 1994; Nagase, 1996).Ethanol exposure alters the balance between MMPs and TIMPs and leads to the development of fibrosis in the liver (Hernandez-Gea and Friedman 2011), which ischaracterized by an excessive depositionof ECM (Siegmund and Brenner, 2005). Additionally, the ethanol hasalso been demonstrated to alter MMP-2 and MMP-9
activities in other tissues such as isolated animal vascular cells and breast cancer cells (Aye et al., 2004; Cullen et al., 2005).
Although it is known that the change of MMPs activities and TIMPs levels may result in pathologic alterations and that ethanol modifies the MMPsactivity and TIMPs levels, the MMP-2 and MMP-9 activities in the prostate, as well as levels of TIMP-1 and TIMP-2 during chronic ethanol ingestion, have not yet been examined. Such analyses may reveal another facet of the damage caused by ethanol to the prostate gland.
The aim of this study was to evaluate whether chronic low- and high-dose ethanol intakemay to alter MMP-2 and MMP-9 activity and the levels of their tissue inhibitor, TIMP-2 and TIMP-1, in the rat dorsal and lateral prostate.
Material and methods
Animals, experimental groups and diet
Forty UCh (University of Chile) male rats were used for this experiment. The UCh variety are ethanol-preferring rats that display voluntary ethanol intake (Mardones & Segovia-Riquelmi, 1983). There are two UCh rat varieties, UChA and UChB, which exhibit low ethanol consumption (0.2-2 g/kg/day) and high ethanol consumption (4-8 g/kg/day) of 10% (v/v) ethanol solution, respectively (Quintanilla et al., 2006). Twenty adults of each variety, UChA and UChB, weighing between 280-330 g (~90 days old), were obtained from the Department of Anatomy, Bioscience Institute, Campus of Botucatu, UNESP – Univ Estadual Paulista (IBB/UNESP).
The Rats that display low and high-dose ethanol intake, UChA and UChB, respectively, were divided into two sub-groups (n=10⁄group): UChA (ethanol-consuming
rats) and UChAC (water-consuming rats); UChB (ethanol-consuming rats) and UChBC (water-consuming rats).
The ethanol ingestion were measured weekly throughout the experimental period (120 days) using a marked test tube. All rats were housed in individual cages in a temperature- and humidity-controlled room under a 12-h light-dark cycle and had free access to filtered tap water and were fed with standard rodent chow Nuvital®(Nuvilab CR-1). The experimental protocols followed the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and were approved by Ethical Committee of the Institute of Bioscience/UNESP (CEEA- Comitê de Ética em Experimentação Animal) Campus of Botucatu, SP, Brazil (IBB/UNESP, Protocol nº 340/2011).
Assessment of gelatinolytic activity of the MMP-2 and MMP-9
MMP-2 and MMP-9 activities were accessed according to the method described by Carvalho et al (2006). The gelatin zymography was measured in the dorsal and lateral prostate tissues from four different rats of each experimental group. The frozen samples were mechanically homogenized in lysis buffer. Then, 35 µg of protein were loaded in SDS-PAGE gels (8%) under non-reducing conditions, containing gelatin at a concentration of 1 mg/ml. Furthermore, purified MMP-2 (20 ng) and MMP-9 (30 pg) (Calbiochem, Boston, MA, USA) were also loaded as positive controls, and molecular weight determinations of MMP-2/MMP-9 were estimated with reference to protein standards (Bio-Rad Laboratories, Inc., Richmond, CA, USA).
Finally, quantitative evaluation of the gelatinolytic activity of captured images was performed by quantifying the lyses bands corresponding to each type of enzyme activity using a computer-based imaging program (NIH freeware Image-J). The values are expressed as the mean ± SD of the totality of IODs for the MMP-2 pro-enzyme, intermediate and active forms and for MMP-9 pro- and active forms.
Measurement of TIMP-1 and TIMP-2
Tissue inhibitors were determined using the commercially available immunoassay kit (Quantikine Rat TIMP-1 and TIMP-2) according to the manufacturer’s instructions (R&D Systems, Inc.). Prostate tissue samples were homogenized and diluted 1:200 (v/v) for TIMP-1 and 1:10 (v/v) for TIMP-2. The concentrations of TIMPs in the samples were determined by extrapolation from an adapted standard curve. TIMPs were determined in the following ranges: TIMP-1 between 37.5 and 2400 pg/ml and TIMP-2 between 1.56and 100 ng/ml. All determinations were performed in duplicate. The coefficient of variation intra-assay was <4% for TIMP-1 and <10% for TIMP-2. The inter-assay value was <8% for TIMP-1 and <12% for TIMP-2.
Statistical analysis
The gelatinolytic activity of the MMP-2 and MMP-9 were evaluated using Student’s t-test. The analysis of TIMP-2 in the lateral prostate of UChA rats and TIMP-1/TIMP-2 in the lateral prostate of the UChB rats was compared using the Mann-Whitney test. The arithmetic mean and standard error medium (Mean ± SEM) were calculated for all the data. Significant differences were set at P< 0.05. The statistical software used was Graph Pad
Instat version 3 (Graph Pad Software, San Diego, CA, USA), and Sigma Plot version 11 (Systat Software, Canada, USA) was used for graphic design.
Results
Ethanol ingestion
The relative ingestion of ethanol was analyzed during all experimental periods, and the results are shown in table 1. Ethanol ingestion by UChA and UChB rats was within the level expected for each variety (Table 1).
Effects of ethanol upon MMP-2 and MMP-9 activity in the dorsal and lateral prostate
Low chronic ethanol did not alter the MMP-9 activity in the dorsal prostate, and MMP-2 activity decreased with ethanol (Fig. 1A). The MMP-2 and MMP-9 activities decreased in the lateral prostate of UChA rats (Fig. 1B).
High chronic ethanol ingestion did not alter the MMP-2 activity in the dorsal prostate, and MMP-9 activity was not observed in the UChB and UChBC groups (Fig. 1C). Similarly, in the lateral prostate of the UChB rats, MMP-2 activity was also not different, but MMP-9 activity was only detectable in the UChBC group. These results indicate that ethanol consumption decreases MMP9 activity in the lateral prostate (Fig. 1D).
TIMP-1 and TIMP-2 in the dorsal and lateral prostate
The TIMP-1 level decreased in the dorsal and lateral prostate of UChA rats (Fig. 2A and B), and the TIMP-2 levels was higher in the lateral prostate of UChA rats (Fig. 2B). In
addition, TIMP-1 and TIMP-2 decreased in UChB rat lateral prostates (Fig. 2D). There were no differences observed in the dorsal prostate (Fig. 2C).
Discussion
The UChA and UChB rats consumed the expected amount of ethanol, which allowed us to evaluate the effects of low and high levels of ethanol ingestion upon MMP-2/MMP-9 activities as well as TIMP-1/TIMP-2 levels in the rat dorsal and lateral prostate.
Low chronic ethanol ingestion (UChA) decreased the MMP-2 activity in dorsal and lateral prostate and the MMP-9 activity in the lateral prostate. The moderate to regular ingestion of ethanol reduces growth of atherosclerotic plaque by reducing MMP-2 and MMP-9 activities, which are responsible for degrading collagen types IV and V surrounding smooth muscle cell (SMC) (Cullen et al., 2005). In this context, moderate ethanol ingestion is important to prevent vascular diseases in humans (Fiotti et al., 2008). Disruption of the MMP/TIMP balance can lead to the excessive accumulation of collagen in the extracellular matrix and consequently result in fibrosis (Hernandez-Gea and Friedman 2011; Surya Narayanan et al., 2011). The decreases of MMPs activities observed in the low-ethanol exposure prostate can lead to prostatic fibrosis that occurs when the matrix synthesis rate exceeds the degradation (Wynn, 2008; Surya Narayanan et al., 2011). Thus, the proteins associated with tissue fibrosis need to be investigated in the lateral prostate of the UChA rats.
The function of TIMPs remains controversial (Brehmer et al., 2003). They are thought to act as a negative regulator for MMP-9 and MMP-2, and when TIMPs levels increase, the MMPs activity decreases. There is evidence that TIMPs, mainly TIMP-2, is a
