Mmp-2 And Mmp-9 activities and Timp-1 and Timp-2 expression in the prostatic tissue of two ethanol-preferring rat models

We investigated whether chronic ethanol intake is capable of altering the MMP-2 and MMP-9 activities and TIMP-2 and TIMP-1 expression in the dorsal and lateral prostatic lobes of low (UChA) and high (UChB) ethanol-preferring rats. MMP-2 and MMP9 activities and TIMP-1 and TIMP-2 expression were significantly reduced in the lateral prostatic lobe of the ethanol drinking animals. Dorsal prostatic lobe was less affected showing no significant alterations in these proteins, except for a reduction in the TIMP-1 expression in UChA rats. These important findings demonstrate that chronic ethanol intake impairs the physiological balance of the prostate extracellular matrix turnover, through downregulation of MMPs, which may contribute to the development of prostatic diseases. Furthermore, since these proteins are also components of prostate secretion, the negative impact of chronic ethanol intake on fertility may also involve reduction of MMPs and TIMPs in the seminal fluid2015COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2011/03394-4; 2011/13713-

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

Orientadores: Sérgio Luis Felisbino, Francisco Eduardo MartinezTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: 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 prostateDoutoradoAnatomiaDoutora em Biologia Celular e Estrutura

Retinol, retinoic acid and its receptors and the rate of cell proliferation/apoptosis in the dorsolateral prostate lobe of adult UCh rats (10% (v/v) ethanol voluntary drinkers)

Orientador: Francisco Eduardo MartinezDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A exposição ao etanol altera a concentração do retinol e do all-trans-ácido retinóico (atAR) em vários tecidos. Os retinóides, retinol e atAR, são importantes para a diferenciação e manutenção das células epiteliais da próstata. O atAR se liga aos receptores de ácido retinóico (RARa, ß e y) e a interação receptor/ligante com a sequência responsiva ao retinóide no DNA, levam à transcrição de genes alvos. Assim, o atAR exerce efeitos no crescimento celular, diferenciação e apoptose, sendo essencial no desenvolvimento e diferenciação de órgãos e tecidos. Nosso objetivo foi analisar o retinol, o ácido retinóico e seus receptores, bem como, o índice de proliferação celular e de apoptose no lobo dorsolateral da próstata de ratos adultos UCh. Os animais foram divididos em quatro grupos experimentais (n=10/grupo): UChA (ingestão voluntária de etanol a 10% (v/v); UChACo (controle - ausência de etanol); UChB (ingestão voluntária de etanol a 10% (v/v) e UChBCo (controle - ausência de etanol). Após 150 dias de experimentação, os animais foram eutanasiados por decapitação e o sangue do tronco e os lobos dorsolaterais das próstatas foram coletados e processados: (1) para análises da concentração do retinol e do atAR no plasma e na próstata por meio de HPLC; (2) e análises de microscopia de luz para a proliferação celular (Ki-67), apoptose (Tunel) e para os receptores de ácido retinóico, por meio dos anticorpos anti-RARa, -ß e -y. O consumo crônico de etanol diminuiu a concentração do retinol no plasma dos grupos UChB (consumo alto de etanol) e UChA (consumo baixo de etanol). A concentração do retinol foi ainda menor no plasma do grupo UChB comparado ao UChA. No entanto, a concentração do retinol no tecido prostático não teve diferença significativa entre os grupos. O atAR aumentou significativamente somente no plasma do grupo UChB. Na próstata, a concentração do atAR aumentou no grupo UChB, enquanto que no UChA não houve diferença estatística. O RAR? na próstata dorsal e lateral dos ratos UCh não foi alterada em função do consumo de etanol. Já os RARß e -? apresentaram aumento do sinal na próstata dorsal do grupo UChB. Não houve diferença no índice de proliferação celular e de apoptose nas próstatas dorsais e laterais dos grupos experimentais. Conclui-se que o etanol altera a concentração do retinol e do atAR no plasma. Essa alteração é diretamente proporcional à quantidade de etanol consumida. Já na próstata, o retinol não é alterado pelo etanol. O consumo alto de etanol altera a concentração do atAR na próstata dorsolateral e a expressão dos RAR ß e y na próstata dorsal. A alteração da expressão dos RAR pode aumentar a sensibilidade da próstata à ação do atAR. O etanol não altera a proliferação celular e a apoptose na próstata dorsal e lateralAbstract: Ethanol exposure alters the concentration of retinol and all-trans retinoic acid (atAR) in several tissues. Retinoids (retinol and atAR) are essential for the differentiation and homeostasis of the prostate epithelial cells. atAR binds to retinoic acid receptors (RAR a, ß and ?) and the interaction receptor/ligand with the sequence responsive to retinoid into DNA lead to transactivation of target genes. Thus, atAR directly produces their effects on cell growth, differentiation and apoptosis. This study aimed to analyze the retinol and all-trans-retinoic acid concentrations and its atAR receptors as well as the cell proliferation and apoptosis index upon the dorsolateral prostate lobe of adult UCh rats. All animals were divided into four experimental groups (n = 10/group): UChA (10% ethanol (v / v) voluntary intake); UChACo (without ethanol consumption); UChB (10% ethanol (v / v) voluntary intake) and UChBCo (without ethanol consumption). After 150 days of experimentation, animals were sacrificed followed by decapitation and trunk blood and dorsolateral prostate lobes collected. Samples of plasma and prostate by concentration analysis of the retinol and atAR were processed for HPLC. The cell proliferation and apoptosis immunoreactivities were assessed by Ki-67 and Tunel, respectively, and nuclear receptors by anti-RAR a,-ß and-y. Chronic ethanol consumption reduced the concentration of plasma retinol in UChB (high ethanol intake) and UChA groups (low ethanol intake). The retinol concentration in plasma was even lower in UChB compared to UChA group. However, the retinol concentration in prostate tissue was not significantly different between the groups. Concentration of atAR increased in plasma of UChB group, and was 96% higher in the UChA group. The prostate, atAR increased in the UChB group, while in UChA group no statistical difference. There was no statistical difference in proliferation cell and apoptosis in the dorsal and lateral prostate lobes between the groups. The expression of RAR a in the dorsal and lateral prostate of UCh rats was not altered as a function of ethanol consumption. Already RAR ß and-y showed increased signal in the dorsal prostate UChB group. We conclude that ethanol alters the concentration of retinol and atAR in plasma. This change is directly proportional to the amount of ethanol consumed. In the prostate, retinol is not altered by ethanol. The high ethanol intake alters the concentration of atAR in dorsolateral prostate and the expression of RARß and RARy in the dorsal prostate. Alteration in expression of RAR can increase sensitivity to the action of the atAR in prostate. Ethanol does not alter cell proliferation and apoptosis in the dorsolateral prostateMestradoAnatomiaMestre em Biologia Celular e Estrutura

Ethanol modulates the synthesis and catabolism of retinoic acid in the rat prostate

All-trans retinoic acid (atRA) maintains physiological stability of the prostate, and we reported 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 strains) on atRA metabolism in the dorsal and lateral prostate. Aldehyde dehydrogenase (ALDH) subtype 1A3 was 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. atRA levels 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 total CYP was increased in the UChA lateral prostate. Ethanol modulates the synthesis and catabolism of atRA in the prostate in a concentration-dependent manner. (C) 2015 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

MMP-2 and MMP-9 activities and TIMP-1 and TIMP-2 expression in the prostatic tissue of two ethanol-preferring rat models

We investigated whether chronic ethanol intake is capable of altering the MMP-2 and MMP-9 activities and TIMP-2 and TIMP-1 expression in the dorsal and lateral prostatic lobes of low (UChA) and high (UChB) ethanol-preferring rats. MMP-2 and MMP-9 activities and TIMP-1 and TIMP-2 expression were significantly reduced in the lateral prostatic lobe of the ethanol drinking animals. Dorsal prostatic lobe was less affected showing no significant alterations in these proteins, except for a reduction in the TIMP-1 expression in UChA rats. These important findings demonstrate that chronic ethanol intake impairs the physiological balance of the prostate extracellular matrix turnover, through downregulation of MMPs, which may contribute to the development of prostatic diseases. Furthermore, since these proteins are also components of prostate secretion, the negative impact of chronic ethanol intake on fertility may also involve reduction of MMPs and TIMPs in the seminal fluid.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Apoptosis of Purkinje and Granular Cells of the Cerebellum Following Chronic Ethanol Intake

Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10 % (<2 g ethanol/kg body weight/day). At 120 days old, rats were anesthetized and decapitated, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), caspase-3, X-linked inhibitor of apoptosis protein (XIAP), and insulin-like growth factor 1-receptor (IGF-1R); real-time PCR (RT-PCR) to determine caspase-3, XIAP, and IGF-1R gene expression; and transmission electron microscopy (TEM). We identified fragmentation of DNA and an increase in caspase-3 protein and XIAP in Purkinje cells, whereas granule cells exhibited increased caspase-3 and XIAP. IGF-1R expression was unchanged. There was no significant difference in gene expression of caspase-3, XIAP, and IGF-1R. There were an increase in lipid droplets, a reduction in the cellular cytoplasm in electrondense nuclei, and changes in the myelin sheath in the cerebellar cortex. In conclusion, our data demonstrated that ethanol induced apoptosis in the Purkinje and granule cells of the cerebellum of adult UChA rats.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP