Colon cancer is the third most common cancer in the United States. However, evidence indicates that a proper diet abundant in fruits and vegetables may be protective against colon cancer development. Bioactive compounds in fruits and vegetables, such as flavonoids and limonoids, have been shown to possess anti-proliferative and antitumorigenic effects in various in vitro and in vivo models of cancer. Since there are few animal studies involving flavonoids and limonoids and colon cancer, this experiment investigated the potentially protective effects of four citrus flavonoids and one limonoid mixture against the promotion stage of chemically-induced colon cancer in rats. Male SD rats (n =60; 10 rats/group) were assigned to receive diets containing 0.1% apigenin, 0.02% naringenin, 0.1% hesperidin, 0.01% nobiletin, 0.035% limonin glucoside/obacunone glucoside mixture, or a control diet (0% flavonoid/limonoid). Rats received the diets for 10 wk and were injected with azoxymethane (15 mg/kg) at wk 3 and 4. The excised colons were evaluated for aberrant crypt foci (ACF) formation, cell proliferation (PCNA assay), apoptosis (TUNEL assay), and iNOS and COX-2 expression. When compared to the control diet, apigenin lowered the number of high multiplicity ACF (> 4 AC/focus) by 57% (P<0.05) and tended to lower the proliferative index (28%; P=0.07), while naringenin lowered both the number of high multiplicity ACF by 51% (P<0.05) and the proliferative index by 32% (P<0.05). Both apigenin and naringenin increased apoptosis of surface colon cells (78% and 97%, respectively; P<0.05) when compared to control diet. Hesperidin, nobiletin, and the limoninglucoside/obacunone glucoside mixture did not have any effects on the above variables measured in this model of colon carcinogenesis. The colonic mucosal protein levels of iNOS or COX-2 were not different among the six diet groups. Evidence suggests that high multiplicity ACF are indicative of future tumor development in both humans and rats. Furthermore, dysregulated proliferation and apoptosis may also lead to tumorigenesis. Therefore, the ability of dietary apigenin and naringenin to reduce high multiplicity ACF, lower proliferation, and increase apoptosis may contribute toward colon cancer prevention. However, their protection is not due to their influence on iNOS and COX-2 protein levels.

Orientador: Luís Fernando Barbisan / Resumo: A capsaicina (8-metil-N-vanilil-trans-6-nonamida) é uma substância alcaloide de natureza lipofílica, responsável pela pungência de pimentas e pimentões. Apresenta propriedades anti-inflamatória, antimicrobiana e antioxidante bem descritas na literatura científica. Assim, este projeto teve como objetivo investigar se a capsaicina apresenta efeito quimioprotetor quando administrada na etapa de promoção/progressão da carcinogênese de cólon induzida pela 1,2-dimetilhidrazina (DMH). Ratos Wistar machos foram alocados em seis grupos experimentais com 10 a 15 animais cada. Os animais dos grupos 1-3 receberam quatro injeções subcutâneas (s.c) do carcinógeno DMH (40mg/kg, duas doses/semana) e os animais do grupo 4-6 receberam quatro injeções s.c de solução de EDTA .Os grupos 2 e 4 receberam por gavagem 5mg/kg p.c e os grupos 3 e 5 receberam 50mg/kg p.c de capsaicina diluída em óleo de milho até o final da 24ª semana do experimento. Após a eutanásia dos animais, os cólons distal, medial e proximal foram corados com azul de metileno a 2% para detecção de focos de criptas aberrantes (FCA). Após a análise de FCAs, os cólons foram processados para análise histológica e classificação de tumores. As amostras congeladas foram utilizadas para análise de expressão de gênica pela técnica Taqman® Low Density Array (TLDA). Os níveis séricos de ALT (p=0,346) e creatinina (p=0,854) foram semelhantes entre os grupos, mostrando que as doses de capsaicina utilizadas não apresentaram ação citotóxica par... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Capsaicin (8-Methyl-N-vanillyl-(trans)-6-nonenamide) is a lipophilic alkaloid, responsible for the pungency in pepper. Its antiinflamatory, antimicrobial and antioxidant properties are well described in the scientific literature. Thus, the objective of this study was to investigate whether capsaicin exerts a chemoprotective effect when administered during 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis promotion/progression in rates. Male Wistar rats were allocated into six groups of 10-15 animals each. Groups 1-3 received 4 subcutaneous injections of DMH (40 mg/kg bw, 2 doses/week), while groups 4-6 received EDTA solution (DMH vehicle, 2 doses/week), followed by intragastric 5mg/kg bw capsaicin diluted in corn oil (G2, G4) or 50mg/kg bw (G3, G5) for 24 weeks (3 doses/week). After sacrifice, blood samples were drawn by heart puncture for the assessment of serum alanine aminotransferase (ALT) and creatinine levels, and the colon was removed. A segment of distal colon and colon tumor samples were frozen in liquid nitrogen at -80ºC. Distal, medial and proximal colon samples were stained with 2% methylene blue for the detection of aberrant crypt foci (ACF).After ACF analysis, colon tumors were processed for histological analysis and tumor classification. The frozen samples were used for the analysis of gene expression by Taqman® Low Density Array (TLDA). Serum ALT (p=0.346) and creatinine (p=0.854) levels were similar among groups, indicating that the capsaicin doses use... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:27:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-29Bitstream added on 2014-06-13T18:56:50Z : No. of bitstreams: 1 silva_frm_me_botfm.pdf: 2481576 bytes, checksum: 0cc379f4b864c599808f845e21be54a1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente estudo foi delineado para investigar a possível ação quimioprotetora do quelato de zinco (ZnGly), na etapa de iniciação da carcinogênese do cólon, induzida pela 1,2- dimetilhidrazina (DMH) em ratos Wistar machos. Os animais foram distribuídos em quatro grupos experimentais e receberam três doses de 40mg/Kg, via subcutânea (sc) de DMH ou EDTA (veiculo da DMH) e 15mg/Kg de quelato de zinco (ZnGly) por gavagem durante quatro semanas. Após a narcose foram coletadas amostras de sangue e, em seguida fragmentos do cólon para dosagens de zinco. O cólon foi removido inteiro, fixado em formalina tamponada a 10%, corado com azul de metileno para análise de incidência de focos de criptas aberrantes (FCAs) e processado histologicamente para análise dos índices de proliferação celular e apoptose. A exposição à DMH aumentou significativamente os índices de proliferação celular e apoptose na mucosa do cólon. A administração do quelato de zinco (ZnGly) não alterou o número e multiplicidade de FCAs e não modificou as taxas de proliferação celular e de apoptose da mucosa do cólon, tanto no grupo tratado com DMH como no respectivo grupo controle. Os resultados deste estudo indicam o zinco não mostrou ação protetora na etapa de iniciação da carcinogênese do cólon induzida pela DMH em ratos Wistar machos / The present study was designed to investigate the possible chemopreventive action of zinc gluconate (ZnGly) on the initiation step of colon carcinogenesis induced by 1,2- dimethylhydrazine (DMH) in male Wistar rats. The animals were divided into four groups and received three doses of DMH (40 mg/Kg, s.c) or EDTA (DMH vehicle) and 15 mg/Kg of zinc glycine (ZnGly) administered by gavage for four weeks. After the narcosis, blood and colon fragments samples were collected for zinc dosage. The entire colon was removed, fixed in buffered formalin 10%, stained with methylene blue for the analysis of ACF formation. The rates of cell proliferation and apoptosis were also analyzed in epithelial crypt cells. The exposure to DMH significantly increased the rates of cell proliferation and apoptosis in epithelial cript cells. Treatment with zinc glycine (ZnGly) did not alter the number and multiplicity of ACFs, as well as apoptosis and cell proliferation indices in both DMH-treated and control group. The present findings indicate that zinc did not present chemopreventive effects on the initiation step of DMH-induced colon carcinogenesis in male Wistar rats

Resumo: O presente estudo foi delineado para investigar a possível ação quimioprotetora do quelato de zinco (ZnGly), na etapa de iniciação da carcinogênese do cólon, induzida pela 1,2- dimetilhidrazina (DMH) em ratos Wistar machos. Os animais foram distribuídos em quatro grupos experimentais e receberam três doses de 40mg/Kg, via subcutânea (sc) de DMH ou EDTA (veiculo da DMH) e 15mg/Kg de quelato de zinco (ZnGly) por gavagem durante quatro semanas. Após a narcose foram coletadas amostras de sangue e, em seguida fragmentos do cólon para dosagens de zinco. O cólon foi removido inteiro, fixado em formalina tamponada a 10%, corado com azul de metileno para análise de incidência de focos de criptas aberrantes (FCAs) e processado histologicamente para análise dos índices de proliferação celular e apoptose. A exposição à DMH aumentou significativamente os índices de proliferação celular e apoptose na mucosa do cólon. A administração do quelato de zinco (ZnGly) não alterou o número e multiplicidade de FCAs e não modificou as taxas de proliferação celular e de apoptose da mucosa do cólon, tanto no grupo tratado com DMH como no respectivo grupo controle. Os resultados deste estudo indicam o zinco não mostrou ação protetora na etapa de iniciação da carcinogênese do cólon induzida pela DMH em ratos Wistar machos / Abstract: The present study was designed to investigate the possible chemopreventive action of zinc gluconate (ZnGly) on the initiation step of colon carcinogenesis induced by 1,2- dimethylhydrazine (DMH) in male Wistar rats. The animals were divided into four groups and received three doses of DMH (40 mg/Kg, s.c) or EDTA (DMH vehicle) and 15 mg/Kg of zinc glycine (ZnGly) administered by gavage for four weeks. After the narcosis, blood and colon fragments samples were collected for zinc dosage. The entire colon was removed, fixed in buffered formalin 10%, stained with methylene blue for the analysis of ACF formation. The rates of cell proliferation and apoptosis were also analyzed in epithelial crypt cells. The exposure to DMH significantly increased the rates of cell proliferation and apoptosis in epithelial cript cells. Treatment with zinc glycine (ZnGly) did not alter the number and multiplicity of ACFs, as well as apoptosis and cell proliferation indices in both DMH-treated and control group. The present findings indicate that zinc did not present chemopreventive effects on the initiation step of DMH-induced colon carcinogenesis in male Wistar rats / Orientador: Maria Aparecida Marchesan Rodrigues / Coorientador: Luis Fernando Barbisan / Banca: Daniel Araki Ribeiro / Banca: Alaor Aparecido Almeida / Mestre

Exposure to polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene are important risk factors for cancer. Three UDP-glucuronosyltransferases, UGT1A9, UGT1A10, and UGT2B7, have been shown to play an important role in the phase II metabolism of carcinogenic metabolites of BaP. Because UGT1A9 and UGT2B7 are well-expressed in digestive tract tissues including liver and colon, it is possible that genetic variations in either enzyme may play an important role in colon cancer risk. However, UGT1A10 is extrahepatic and is expressed in the oral cavity and the larynx; therefore, genetic variations in this enzyme may play an important role in risk for orolaryngeal cancer. This study examined UGT1A9-, UGT1A10-, and UGT2B7-specific sequences for polymorphisms that play a role in cancer susceptibility. For the UGT1A9 gene, two missense polymorphisms at codons 167 (Val>Ala) and 183 (Cys>Gly) were identified. A previously-reported missense polymorphism was identified for the UGT2B7 gene. To assess the potential role of UGT1A10 variants as a risk factor for orolaryngeal cancer, PCR-RFLP was used to identify UGT1A10 genotypes in DNA specimens isolated from 115 African American newly-diagnosed orolaryngeal cancer cases and 115 non-cancer controls individually matched by age and race. A significantly decreased risk for orolaryngeal cancer was observed for subjects possessing one or more UGT1A10139Lys alleles as determined by crude analysis or after logistic regression analysis adjusting for age, sex, smoking and alcohol consumption. These results strongly suggest that the UGT1A10139Lys polymorphism may play an important protective role in risk for orolaryngeal cancer. To determine whether the change in amino acid sequence at codon 183 results in aberrant UGT1A9 enzyme activity, functional characterization of the wild-type- and variant-encoded UGT1A9 isoforms was performed in vitro. Cell homogenates were prepared from UGT1A9-transfected HK293 cells and glucuronidation assays were performed against various carcinogens/carcinogen metabolites. A significant (p<0.001) 3- to 4-fold decrease in enzyme activity, determined by HPLC analysis, was observed for the UGT1A9183Gly variant as compared to its wild-type counterpart for all substrates analyzed. These results demonstrate that the UGT1A9 (Cys183Gly) polymorphism significantly alters UGT1A9 function and could potentially play an important role as risk modifier for digestive tract cancers.

Das Selenoprotein Glutathionperoxidase 2 (GPx2) ist ein epithelzellspezifisches, Hydroperoxide-reduzierendes Enzym, welches im Darmepithel, vor allem in den proliferierenden Zellen des Kryptengrundes, exprimiert wird. Die Aufrechterhaltung der GPx2-Expression im Kryptengrund auch bei subadäquatem Selenstatus könnte darauf hinweisen, dass sie hier besonders wichtige Funktionen wahrnimmt. Tatsächlich weisen GPx2 knockout (KO)-Mäuse eine erhöhte Apoptoserate im Kryptengrund auf. Ein Ziel dieser Arbeit war es deshalb, die physiologische Funktion der GPx2 näher zu untersuchen. In Kryptengrundepithelzellen aus dem Colon selenarmer GPx2 KO-Mäuse wurde eine erhöhte Caspase 3/7-Aktivität im Vergleich zum Wildtyp (WT) festgestellt. Zudem wiesen diese Zellen eine erhöhte Suszeptibilität für oxidativen Stress auf. Die GPx2 gewährleistet also den Schutz der proliferierenden Zellen des Kryptengrundes auch bei subadäquater Selenversorgung. Des Weiteren wurde im Colon selenarmer (-Se) und -adäquater (+Se) GPx2 KO-Mäuse im Vergleich zum WT eine erhöhte Tumornekrosefaktor α-Expression und eine erhöhte Infiltration von Makrophagen festgestellt. Durch Fütterung einer selensupplementierten Diät (++Se) konnte dies verhindert werden. In GPx2 KO-Mäusen liegt demnach bereits basal eine niedriggradige Entzündung vor. Dies unterstreicht, dass GPx2 vor allem eine wichtige antiinflammatorische Funktion im Darmepithel besitzt. Dem Mikronährstoff Selen werden protektive Funktionen in der Colonkanzerogenese zugeschrieben. In einem Mausmodell der Colitis-assoziierten Colonkanzerogenese wirkte GPx2 antiinflammatorisch und hemmte so die Tumorentstehung. Auf der anderen Seite wurden jedoch auch prokanzerogene Eigenschaften der GPx2 aufgedeckt. Deshalb sollte in dieser Arbeit untersucht werden, welchen Effekt ein GPx2 knockout in einem Modell der sporadischen, durch Azoxymethan (AOM) induzierten, Colonkanzerogenese hat. Im WT kam es in präneoplastischen Läsionen häufig zu einer erhöhten GPx2-Expression im Vergleich zur normalen Darmmucosa. Eine derartige Steigerung der GPx2-Expression wurde auch in der humanen Colonkanzerogenese beschrieben. Das Fehlen der GPx2 resultierte in einer verminderten Entstehung von Tumoren (-Se und ++Se) und präneoplastischen Läsionen (-Se und +Se). Somit förderte GPx2 die Tumorentstehung im AOM-Modell. Acht Stunden nach AOM-Gabe war im GPx2 KO-Colon im Vergleich zum WT eine erhöhte Apoptoserate in der Kryptenmitte (-Se, +Se), nicht jedoch im Kryptengrund oder in der ++Se-Gruppe zu beobachten. Möglicherweise wirkte GPx2 prokanzerogen, indem sie die effiziente Elimination geschädigter Zellen in der Tumorinitiationsphase verhinderte. Eine ähnliche Wirkung wäre auch durch die erhöhte GPx2-Expression in der Promotionsphase denkbar. So könnte GPx2 proliferierende präneoplastische Zellen vor oxidativem Stress, Apoptosen, oder auch der Antitumorimmunität schützen. Dies könnte durch ein Zusammenwirken mit anderen Selenoproteinen wie GPx1 und Thioredoxinreduktasen, für die ebenfalls auch prokanzerogene Funktionen beschrieben wurden, verstärkt werden. Eine wichtige Rolle könnte hier die Modulation des Redoxstatus in Tumorzellen spielen. Die Variation des Selengehalts der Diät hatte im WT einen eher U-förmigen Effekt. So traten in der –Se und ++Se-Gruppe tendenziell mehr und größere Tumore auf, als in der +Se Gruppe. Zusammenfassend schützt GPx2 also die proliferierenden Zellen des Kryptengrundes. Sie könnte jedoch auch proliferierende transformierte Zellen schützen und so die sporadische, AOM-induzierte Colonkanzerogenese fördern. In einem Modell der Colitis-assoziierten Colonkanzerogenese hatte GPx2 auf Grund ihrer antiinflammatorischen Wirkung einen gegenteiligen Effekt und hemmte die Tumorentstehung. Die Rolle der GPx2 in der Colonkanzerogenese ist also abhängig vom zugrunde liegenden Mechanismus und wird maßgeblich von der Beteiligung einer Entzündung bestimmt. / The selenoprotein glutathione peroxidase 2 (GPx2) is a hydroperoxide-reducing enzyme that is mainly expressed in the gastrointestinal epithelium, especially in the crypt base were the proliferating cells reside. GPx2 expression is maintained even when the selenium supply is limited, which indicates that GPx2 might have an important function in these cells. Indeed, GPx2 knockout (KO)-mice have an enhanced rate of apoptosis in the crypt base. Therefore one aim of this study was to further elucidate the physiological function of the GPx2. Isolated colonic crypt base epithelial cells of selenium deficient GPx2 KO-mice were found to have a higher caspase 3/7 activity than wild type (wt) cells. Moreover they exhibited an enhanced susceptibility for oxidative stress. Thus GPx2 protects the proliferative crypt base cells of the intestine, especially when the selenium supply is limited. Additionally an enhanced expression of tumor necrosis factor α and an enhanced infiltration of macrophages were detected in the colon of GPx2 KO-mice in comparison to the wt. These effects were observed on a selenium deficient (-Se) and -adequate (+Se) diet, but could be prevented by feeding a selenium supplemented (++Se) diet. Accordingly, GPx2 KO-mice have a basal low grade inflammation. This underlines, that GPx2 has an important anti-inflammatory function in the intestinal epithelium. Selenium deficiency is linked to an increased risk of developing colorectal cancer. In a mouse model of colitis-associated colon carcinogenesis, GPx2 had anti-inflammatory and thus anticarcinogenic effects. However, also procarcinogenic functions of the GPx2 have been observed. Therefore, this study aimed to analyse the role of GPx2 in a model of non-inflammation triggered, sporadic colon carcinogenesis induced by azoxymethane (AOM). In preneoplastic lesions of wt mice, an enhanced expression of GPx2 in comparison to the normal mucosa was frequently observed. An upregulation of GPx2 expression has also been described in human colon carcinogenesis. GPx2 KO mice had less tumors (-Se and ++Se) and less preneoplastic lesions (-Se, +Se) than wt mice. Accordingly GPx2 promotes colon carcinogenesis in the AOM-model. Eight hours after AOM-application, a higher rate of apoptosis was observed in the mid-crypt region of the colon of GPx2 ko mice in comparison to wt mice in the –Se and +Se groups, but not in the ++Se group or in the crypt base. Thus GPx2 might act procarcinogenic by preventing the elimination of cells with DNA-damage in the tumor initiation stage. Similarly, the enhanced GPx2-expression in preneoplastic cells could promote tumorigenesis by protecting these cells from oxidative stress, apoptosis or antitumor immunity. This effect might be enhanced by other selenoproteins like GPx1 or thioredoxin reductases that have also been reported to possess procarcinogenic properties and it might be closely related to the regulation of the redox state of tumor cells. In wt mice, the selenium content of the diet turned out to have a rather U-shaped effect on colon carcinogenesis. In the –Se and ++Se groups, wt mice tended to have more and larger tumors than in the ++Se group. In conclusion, GPx2 protects the proliferating cells of the intestinal crypt base, but it could also protect proliferating transformed cells and thus promote sporadic, AOM-induced colon carcinogenesis. In contrast, GPx2 acted anticarcinogenic in a model of colitis-associated colon carcinogenesis due to its antiinflammatory properties. Thus, the role of GPx2 in colon carcinogenesis depends on the underlying mechanisms, especially on the involvement of an inflammation.

Submitted by Nelci Antunes de Moura (nelcimoura@gmail.com) on 2016-05-20T00:29:22Z No. of bitstreams: 1 tese Nelci final.pdf: 3449766 bytes, checksum: 82850e9746cb84286bf3b3204344f1e9 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-23T19:39:07Z (GMT) No. of bitstreams: 1 moura_na_dr_bot.pdf: 3449766 bytes, checksum: 82850e9746cb84286bf3b3204344f1e9 (MD5) / Made available in DSpace on 2016-05-23T19:39:07Z (GMT). No. of bitstreams: 1 moura_na_dr_bot.pdf: 3449766 bytes, checksum: 82850e9746cb84286bf3b3204344f1e9 (MD5) Previous issue date: 2015-12-18 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O ferro heme presente na carne vermelha está associado ao aumento da incidência do câncer colorretal (CCR). O heme pode catalisar a formação de compostos nitrosos e a peroxidação lipídica no lúmen intestinal. No entanto, os efeitos pró-carcinogênicos do heme podem ser inibidos por alguns compostos como os sais de cálcio, clorofila entre outros. Sabe-se que o indol-3-carbinol (I3C), presente nas plantas da família das Brassicas e os simbióticos são compostos promissores na prevenção do câncer de cólon, atuando em via de proliferação, apoptose e modulação da microbiota intestinal. Dessa forma, o objetivo desse estudo foi o de avaliar os efeitos da ingestão de simbiótico (prebiótico inulina associado ao probiótico Bifidobacterium lactis bb-12) e de I3C, isolados ou em associação sobre o processo de carcinogênese de cólon induzido pela 1,2-dimetilhidrazina (DMH) em ratos Wistar alimentados ou não com dieta suplementada com heme. Os animais foram alocados em 9 grupos, os grupos 1 a 8 (n=12) receberam quatro doses de DMH (40 mg/Kg) nas duas semanas iniciais do experimento. Os grupos 1 e 9 (n=12 e 5) receberam ração basal até o final do experimento e os grupos 2 a 8 receberam ração basal suplementada com heme, heme+I3C, heme+simbiótico, heme+I3C+simbiótico, I3C, simbiótico e I3C+simbiótico, respectivamente. A eutanásia ocorreu ao final da 25ª semana. Neste momento foi realizada a coleta do cólon com os respectivos tumores e amostras de fezes do ceco. Em seguida, procedeu-se a medida dos tumores e coleta de amostras para biologia molecular. Após a fixação em formalina tamponada e a retirada dos tumores, realizou-se a contagem de focos de criptas aberrantes (FCA) pela coloração de azul de metileno. Realizou-se a análise histológica dos tumores e a análise da expressão de 95 genes relacionados a via da carcinogênese colônica, pela técnica Taqman Low Density Array, e a expressão proteica da E-caderina, TGFB1 (Transforming growth factor beta 1) e RAF1 (Serine/threonine-protein kinase) por Western Blotting. Foram analisados os índices de proliferação celular e apoptose pelo PCNA (Proliferating cell nuclear antigen) e caspase 3-clivada, respectivamente, tanto nos cólons como em tumores, e a expressão de β-catenina e E-caderina nos tumores, por imunoistoquímica. Células da linhagem Caco-2 foram incubadas com água fecal extraída das fezes do ceco e submetidas a testes de citotoxicidade e genotoxidade pelos testes do MTT (mitochondrial tetrazolium test) e Cometa, respectivamente. Os dados foram comparados utilizando-se o software Sigma Stat 3.5 e Expression Suíte para expressão gênica. Foi observado aumento significativo no número de criptas aberrantes (CA) no grupo que recebeu heme (G2) quando comparado ao grupo que recebeu apenas ração basal (G1). Redução significativa no número de CA foi observada no grupo que recebeu heme+I3C (G3) e heme+simbiótico (G4) quando comparado ao grupo que recebeu heme (G2). O número de FCA totais com ≥ 9 criptas aberrantes foi significativamente menor no grupo que recebeu heme+simbiótico (G4) quando comparado ao grupo que recebeu heme (G2). Entretanto, aumento significativo no número de tumores com mais de 60 mm3 foi observado no grupo suplementado com heme+I3C+simbiótico (G5), quando comparado ao grupo que recebeu heme (G2). Além disso, foi observado aumento significativo na incidência de tumores invasivos no grupo que recebeu heme+I3C+simbiótico (G5) quando comparado ao grupo que recebeu heme (G2). Os tumores do grupo suplementado com heme+I3C+simbiótico (G5) apresentaram baixa expressão dos genes Cdh1, Tgfb1, Appl1 e alta expressão do Raf1, já os tumores do grupo suplementado com heme +I3C (G3) apresentaram baixa expressão do Cdh1. A água fecal do grupo que recebeu heme (G2) apresentou significativamente maior citotoxicidade e genotoxicidade quando comparado ao grupo que recebeu ração basal (G1). Com relação aos tratamentos, a água fecal do grupo que recebeu heme+I3C (G3) e heme e simbiótico (G4) apresentaram água fecal significativamente com menor potencial genotóxico quando comparada ao grupo que recebeu heme (G2). No entanto, o grupo que recebeu heme+I3C+simbiótico (G5) apresentou aumento significativo na genotoxicidade da água fecal. Dessa forma, concluímos que o heme associado a uma dieta com níveis normais de cálcio não é um potente indutor de FCA, mas aumenta a citotoxicidade e genotoxicidade da água fecal. No entanto, tanto o I3C como o simbiótico reduzem os efeitos citotóxicos/genotóxicos da ingestão de heme. Contudo, a associação do heme+I3C+simbiótico apresentou efeito promotor da carcinogênese de cólon. / Colorectal cancer (CRC) is the third most common type of cancer worldwide. Hemin iron, which is found in red meat, catalyzes the formation of carcinogenic N-nitroso compounds and lipid peroxidation end-products in the colon lumen. The procarcinogenic effect of hemin is known to be inhibited by molecules, such as calcium, chlorophyll and others. However, the preventive effect of indole 3-carbinol and synbiotics on colon carcinogenesis remains uninvestigated. The aim of this study was to assess the modifying effects of a synbiotic (inulin+ Bifidobacterium lactis) and/or I3C against dimethylhidrazine (DMH)-induced colon carcinogenesis in hemin-fed male Wistar rats. Nine groups of animals were evaluated. Groups 1–8 received a total of four s.c. DMH injections (40 mg/kg b.w.) over 2 weeks, whereas group 9 was given EDTA solution (vehicle). Two weeks after DMH-initiation, G1 and G9 were fed a basal diet while groups G2, G3, G4, G5, G6, G7 and G8 received a basal diet containing hemin, hemin+I3C, hemin+synbiotic, hemin+I3C+synbiotic, I3C, synbiotic and I3C+synbiotic, respectively, during 23 weeks. At 25 week, all animals were killed and their colons were removed. Cecal contents were collected to determine fecal water cytotoxicity and genotoxicity (DNA damage) in Caco-2 cells. Colon tumors were measured and samples were collected and stored at -800C. The colons were fixed flat in 10% buffered formalin for 24 h and stained with 1.0% methylene blue for classical ACF analysis and quantification. Tumor incidence and multiplicity were assessed after histopathological analysis. Gene and protein expression were determined in tumor samples alone. The total number of aberrant crypts (AC) was significantly higher (p= 0.03) in the hemin group (G2) than in the group fed basal diet (G1). AC number in both hemin+I3C (G3) and hemin+synbiotic (G4) groups was also significantly lower than in the group fed hemin (G2). Tumor volume was higher in the hemin+I3C+ synbiotic (G5) group and invasive adenocarcinoma was more frequent in the hemin+I3C+synbiotic group (G5) than in the group fed hemin (G2). Colon tumor expression analysis showed that in comparison with the group fed hemin (G2), Cdh1, Tgfb1 and Appl1 were downregulated while Raf1 was upregulated in the group hemin+I3C+synbiotic (G5), and Cdh1 was down-regulated in the group hemin+I3C (G3). Fecal water cytotoxicity in the hemin group (G2) was higher than in groups fed basal diet (G1) and hemin+I3C (G3). Fecal water genotoxicity was also significantly higher in the group fed hemin alone (G2) than in the basal diet group (G1), as well as, in groups fed hemin+I3C (G3) and hemin+synbiotics (G4). However, when compared to hemin alone (G2), fecal water from group hemin+I3C+ synbiotics (G5) presented the highest DNA damage levels. Our results suggest that although hemin in a regular-calcium diet was not a powerful ACF promoter, it increased fecal water citotoxicity and genotoxicity. On the other hand, hemin associated with either I3C or synbiotics prevented ACF promotion. Nonetheless, a synergistic interaction among hemin, I3C and synbiotic did promote DMH-induced tumorigenesis. / FAPESP: 2011/23699-4

Orientador: Luís Fernando Barbisan / Resumo: O ferro heme presente na carne vermelha está associado ao aumento da incidência do câncer colorretal (CCR). O heme pode catalisar a formação de compostos nitrosos e a peroxidação lipídica no lúmen intestinal. No entanto, os efeitos pró-carcinogênicos do heme podem ser inibidos por alguns compostos como os sais de cálcio, clorofila entre outros. Sabe-se que o indol-3-carbinol (I3C), presente nas plantas da família das Brassicas e os simbióticos são compostos promissores na prevenção do câncer de cólon, atuando em via de proliferação, apoptose e modulação da microbiota intestinal. Dessa forma, o objetivo desse estudo foi o de avaliar os efeitos da ingestão de simbiótico (prebiótico inulina associado ao probiótico Bifidobacterium lactis bb-12) e de I3C, isolados ou em associação sobre o processo de carcinogênese de cólon induzido pela 1,2-dimetilhidrazina (DMH) em ratos Wistar alimentados ou não com dieta suplementada com heme. Os animais foram alocados em 9 grupos, os grupos 1 a 8 (n=12) receberam quatro doses de DMH (40 mg/Kg) nas duas semanas iniciais do experimento. Os grupos 1 e 9 (n=12 e 5) receberam ração basal até o final do experimento e os grupos 2 a 8 receberam ração basal suplementada com heme, heme+I3C, heme+simbiótico, heme+I3C+simbiótico, I3C, simbiótico e I3C+simbiótico, respectivamente. A eutanásia ocorreu ao final da 25ª semana. Neste momento foi realizada a coleta do cólon com os respectivos tumores e amostras de fezes do ceco. Em seguida, procedeu-se a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Colorectal cancer (CRC) is the third most common type of cancer worldwide. Hemin iron, which is found in red meat, catalyzes the formation of carcinogenic N-nitroso compounds and lipid peroxidation end-products in the colon lumen. The procarcinogenic effect of hemin is known to be inhibited by molecules, such as calcium, chlorophyll and others. However, the preventive effect of indole 3-carbinol and synbiotics on colon carcinogenesis remains uninvestigated. The aim of this study was to assess the modifying effects of a synbiotic (inulin+ Bifidobacterium lactis) and/or I3C against dimethylhidrazine (DMH)-induced colon carcinogenesis in hemin-fed male Wistar rats. Nine groups of animals were evaluated. Groups 1–8 received a total of four s.c. DMH injections (40 mg/kg b.w.) over 2 weeks, whereas group 9 was given EDTA solution (vehicle). Two weeks after DMH-initiation, G1 and G9 were fed a basal diet while groups G2, G3, G4, G5, G6, G7 and G8 received a basal diet containing hemin, hemin+I3C, hemin+synbiotic, hemin+I3C+synbiotic, I3C, synbiotic and I3C+synbiotic, respectively, during 23 weeks. At 25 week, all animals were killed and their colons were removed. Cecal contents were collected to determine fecal water cytotoxicity and genotoxicity (DNA damage) in Caco-2 cells. Colon tumors were measured and samples were collected and stored at -800C. The colons were fixed flat in 10% buffered formalin for 24 h and stained with 1.0% methylene blue for classical ACF analysis and qua... (Complete abstract click electronic access below) / Doutor

O isoprenóide geraniol (GO) apresentou atividade quimiopreventiva quando administrado continuamente durante as fases de iniciação e pós-iniciação em modelo de carcinogênese experimental de cólon por meio da redução do número de focos de criptas aberrantes (FCAs) totais FCAs &#8805; 4 criptas e aumento de apoptose no cólon distal. Dessa forma, optou-se por avaliar os eventuais efeitos de três doses de GO (GO1: 25mg/100g de peso corpóreo [p.c.], G02: 50 mg/100g de p.c. e G03: 100 mg/100g de p.c.) em categorias de lesões pré-neoplásicas (LPNs) induzidas por dimetilhidrazina (DMH) durante a fase de pós-iniciação tardia de modelo de carcinogênese experimental de cólon, caracterizada por apresentar lesões mais avançadas e com alto grau de alterações celulares morfológicas, bioquímicas e moleculares denominadas de displasia. Para isso, analisamos diferentes biomarcadores como: FCAs totais e FCAs < ou &#8805; 4 criptas em cólons corados com azul de metileno; focos depletados ou positivos de mucina (FPMs ou FDMs) em cólons corados com azul de toluidina; FCAs convencionais ou displásicos por meio de análise histopatológica em cortes corados com hematoxilina e eosina (HE) e focos positivos ou negativos para beta-catenina (FPBCs ou FNBCs) citoplasmática e/ou nuclear por meio de imunoistoquímica. Além disso, células apoptóticas foram identificadas utilizando-se critérios morfológicos clássicos em FCAs &#8805; 4 no cólon distaI e a expressão de genes envolvidos na carcinogênese de cólon foi avaliada por meio de RT-PCR: HMGCoA-redutase na mucosa colônica e K-Ras e c-myc em FCAs microdissecados. Em relação ao grupo controle, foi possível observar que o grupo tratado com a maior dose de GO (G03) reduziu a freqüência de FCAs &#8805; 4 criptas e FDMs, além de aumentar a apoptose em FCAs &#8805; 4 displásicos no cólon distaI (p &#8804; 0,05). Já, em relação aos outros biomarcadores e às expressões de HMGCoA-redutase, K-Ras e c-myc não observamos diferenças estatísticas entre os tratamentos (p > 0,05). A partir desses resultados, podemos concluir que a dose de 100 mg/100 g de p.c. de GO mostrou ser mais interessante do ponto de vista quimiopreventivo com efeitos observados principalmente no cólon distaI, onde há maiores relatos de incidência de adenocarcinomas colônicos, tanto em animais quanto em humanos. Assim, a indução da morte celular programada em FCAs &#8805; 4 preferencialmente displásicos poderia representar um mecanismo importante de atuação de G03 na redução da freqüência de FCAs &#8805; 4 criptas e de FDMs (também utilizado como marcador de displasia) durante a fase de pós-iniciação tardia de modelo de carcinogênese experimental de cólon. / The isoprenoid geraniol (GO) showed chemopreventive activity when administered continuously during the initiation and post-initiation phases in an experimental model of colon carcinogenesis by reducing the number of total aberrant crypt foci (ACF) and ACFs &#8805; 4 crypts, as well as increasing apoptosis in the distal colon. We therefore chose to evaluate the effects of three different doses of GO (GO1: 25 mg/100 g body weight [b.w.], GO2: 50 mg/100 g b.w. and GO3: 100 mg/100 g of b.w.) on preneoplastic lesions (PNLs) induced by dimethylhydrazine (DMH) during late post-initiation in an experimental model of colon carcinogenesis that is characterized by more advanced lesions and a higher degree of cellular alterations morphological, biochemical and molecular (dysplasia) than previous models. For this study, we analyzed the following biomarkers: total ACFs, ACFs < 4 crypts, and ACFs &#8805; 4 erypts in colons stained with methylene blue; mucin-depleted or mucin-positive foci (MDFs or MPFs) in colons stained with toluidine blue; ACFs, through conventional or dysplastie histopathological analysis of sections stained with hematoxylin and eosin (HE); and cytoplasmic vs. nuclear foci reactivity for beta-catenin (foci positive for beta-eatenin (FPBC) or foci negative for beta catenin (FNBC)) using immunohistochemistry. Additionally, apoptotic cells were identified using classical morphologic criteria in ACFs &#8805; 4 crypts in the distal colon, and the expression of several genes involved in colon carcinogenesis was assessed by RT-PCR, including HMG-CoA reductase in the colonic mucosa and K-Ras and c-myc in microdissected ACFs. Relative to the control group, we observed that the group receiving the highest dose of GO (GO3 group) had a reduced frequency of both ACFs &#8805; 4 crypts and MDFs and that apoptosis increased in dysplastic ACFs &#8805; 4 crypts in the distal colon (p < 0, 05). Expression of HMG-CoA reductase, K-Ras and c-myc did not differ between treatments (p > 0, 05). Based on these results, we conclude that the 100 mg/100 g b.w. dose of GO is the most promising, as it shows evidence of chemopreventive effects mainly in the distal colon, which is a region that is reported to have a higher incidence of colonic adenocarcinomas, both in animaIs and in humans. lnduction of programmed cell death by GO3 in ACFs &#8805; 4 specifically dysplastic could represent an important mechanism of action in reducing the frequency of both ACFs &#8805; 4 crypts and MDFs during late post-initiation in this experimental model of colon carcinogenesis.

Das ITC SFN und der Mikronährstoff Se sind bekannt als chemopräventive Inhaltsstoffe von Gemüse der Brassica-Familie, welcher auch Brokkoli angehört. Die Wirkungen von sowohl SFN als auch Se beruhen auf zahlreichen verschiedenen Mechanismen. Es existieren jedoch Schnittstellen, an welchen Interaktionen beider Substanzen möglich sind. Basierend auf diesem Wissen wurden in dieser Arbeit Wechselwirkungen zwischen SFN und Se auf die Aktivität sowie Expression von Phase II Enzymen und Selenoproteinen untersucht. Der Einfluss der Kombination von SFN und Se auf die unter physiologischen Bedingungen stattfindende Proliferation und Apoptose war ebenso Gegenstand der Arbeit wie die Modulation von Entzündungsprozessen sowie der Tumorentstehung während der entzündungsverstärkten Colonkanzerogenese im Mausmodell. Das hinsichtlich seiner Wirksamkeit mit aus GRA hydrolysiertem SFN zunächst als vergleichbar befundene synthetische SFN wurde für die Untersuchung im AOM/DSS-induzierten Colontumormodell gewählt und in Kombination mit 3 verschiedenen Selendiäten verabreicht. Der Einfluss von SFN und Se auf Phase II Enzyme und Selenoproteine entlang des GIT war organabhängig und nach 4 Wochen geringer als nach 7 Tagen. Die schwächere Induktion deutet auf eine Anpassung des Organismus hin. Ein SFN-vermittelter Effekt auf NQO1 war im Selenmangel am deutlichsten. Die Aktivität des Selenoproteins TrxR wurde hingegen erst bei ausreichender Selenversorgung durch SFN beeinflusst. Die als Nrf2-Zielgen bekannte und in der Hierarchie der Selenoproteine einen hohen Rang einnehmende GPx2 konnte in bestimmten Organen bereits unter selenarmen Bedingungen durch SFN induziert werden. Eine Überexpression des Enzyms war jedoch nicht möglich. SFN steigerte, unabhängig vom Selenstatus, im oberen Abschnitt des GIT und im Colon die Aktivität der GST. Eine Induktion des eigenen Metabolismus wäre somit denkbar. Im Falle eines Mangels an GPx2 wurde GPx1 bei hinreichender Selenversorgung stärker exprimiert, allerdings konnte sie die Funktion von GPx2 nicht völlig erset-zen. Im Selenmangel kann die Aktivitätssteigerung der TrxR im Dünndarm, dem Ab-schnitt der Selenabsorption, als ein Versuch der GPx2-Kompensation angesehen werden. SFN war nicht in der Lage, über eine Aktivierung des Nrf2/ARE-Signalweges kompensatorische Effekte zu induzieren. Apoptotische Prozesse wurden unter physiologischen Bedingungen nur marginal durch SFN und Se moduliert. Das elektrophile ITC konnte lediglich im Selenmangel Apoptose im luminalen Bereich der Colonkrypten induzieren. Die durch supranutritive Selenkonzentration induzierte Apoptose im Kryptengrund wurde nicht durch SFN beeinflusst. Einer bei Abwesenheit der GPx2 erhöhten Apoptoserate im Kryptengrund wirkte SFN bei adäquater Selenversorgung entgegen, war indessen proapoptotisch unter selendefizienten Konditionen. Der Einfluss von SFN auf die Entzündung war deutlich abhängig vom Selenstatus. Während SFN im Selenmangel anscheinend prooxidative Prozesse induzierte und die Entzündungssymptome verschlimmerte, wirkte es unter adäquatem Selenstatus an-tiinflammatorisch. Den vergleichsweise milden Grad der Entzündung im selensupplementierten Status konnte SFN nicht zusätzlich beeinflussen. SFN veränderte die Inzi-denz colorektaler Tumore nicht. Ein, die Tumorinitiation blockierender SFN-Effekt durch direkte Hemmung der metabolischen Aktivierung des Prokanzerogens im selenadäquaten Zustand scheint offensichtlich. Eine Überversorgung mit Se kann protektiv im Hinblick auf Entzündung oder Colonkanzerogenese sein, jedoch bewirkt SFN keinen zusätzlichen Schutz. Kombinationseffekte von SFN und Se in Bezug auf Phase II Enzyme, Selenoproteine und Apoptose sowie die entzündungsverstärkte Colonkanzerogenese sind nicht eindeutiger Natur und können, abhängig vom Endpunkt, synergistische oder antagonistische Züge aufweisen. Eine bei Selendefizienz deutlichere Wirkung von SFN kann mit Hilfe der gesteigerten Aktivierung von Nrf2 erklärt werden, dies muss jedoch nicht von Vorteil sein. Bei adäquater Selenversorgung kann SFN kurzfristig antiinflammatorische und antikanzerogene Prozesse induzieren. Von einer längerfristigen ständigen SFN-Aufnahme in Form von GRA-reichen Brassicacea ist jedoch abzuraten, da von einer Adaptation auszugehen ist. Die Wirkung von SFN innerhalb der komplexen Pflanzenmatrix bleibt Gegenstand zukünftiger Untersuchungen. / Sulforaphane (SFN), a versatile actor derived from broccoli or other brassicaceae, is proposed to be a dietary anticarcinogen. Together with an adequate selenium status, it has been associated with a decreased risk for developing certain forms of cancer. In our mouse model, we investigate the influence of SFN and Se on the expression and activity of selenoproteins and phase II enzymes as well as the effects on inflammation triggered colon carcinogenesis. SFN increased NQO1 activity and protein expression significantly in the ileum, in both, Se-deficiently and Se-adequately fed animals. TrxR activity was increased in Se-adequately compared to Se-deficiently fed mice, SFN positively affected TrxR activity only in the former ones. An increase of GPx2 protein expression by SFN was observed in the ileum of mice of both diets. GPx1 reacts sensitively on Se supply. GST was the only enzyme analyzed being significantly increased by SFN on activity level in the colon. All AOM/DSS treated animals showed an inflammation, which was attenuated by SFN within Se-adequacy. In contrast, Se-deficient animals showed a more severe inflammation. The administration of SFN therefore seemed to enhance this even more and to be not beneficial in this case. SFN inhibited colon carcinogenesis in Se-adequate mice when being administered together with AOM. To summarize, both, GPx2 and TrxR, require selenium in order to be synthesized. In contrast to TrxR, the SFN-mediated induction of GPx2, the highest ranking selenoprotein, does not depend on additional selenium supply. Whereas distinct effects by SFN were observed in the ileum, only GST was influenced by SFN in the colon. SFN seems to induce its own metabolism. In conclusion, SFN and Se attenuate inflammation and colon carcinogenesis, preferably by means of up-regulating the endogenous defense system and inhibiting the metabolic activation of AOM.