Efeito da suplementação da dieta com feijão-frade (Vigna unguiculata) num modelo animal de cancro colorretal
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2020-10-30
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O cancro colorretal (CCR) é uma doença comum. A maioria dos casos diagnosticados são espontâneos e difíceis de prevenir com base na alteração de fatores ambientais e de hábitos alimentares. Comprovada a associação entre a melhoria no estado de saúde individual e redução significativa nas taxas de mortalidade associadas ao cancro pela adesão a certos tipos de dieta, a população saudável e os doentes oncológicos são encorajados a aderir a um padrão dietético semelhante à dieta mediterrânica. Nesta dissertação, pretende-se avaliar os efeitos da suplementação de feijão-frade, leguminosa incluída na dieta mediterrânica, na dieta de um modelo experimental de CCR induzido através da administração sequencial de azoximetano (AOM) e dextran-sulfato de sódio (DSS). Para alcançar estes objetivos utilizaram-se quarenta e oito (n=48) murganhos fêmeas da estirpe FVB/n (Mus musculus), com idade compreendida entre os 5-6 meses, divididos aleatoriamente em quatro grupos: grupo controlo (n=9), grupo induzido (n=13), grupo induzido/20% (m/m) farinha de feijão frade (FFF) (n=12) e grupo induzido/50% (m/m) FFF (n=14). O AOM foi administrado uma única vez por via intraperitoneal (7,5 mg/kg). Uma semana depois, os animais foram expostos na água de bebida a 1,5% de DSS durante 7 dias seguidos em três ciclos, com intervalos de 7 dias entre ciclos. Os animais tiveram acesso ad libitum às respetivas dietas durante 13 semanas e foram observados diariamente para registar o seu estado geral. Durante o ensaio experimental foram registadas semanalmente a temperatura corporal, a massa corporal, o consumo de alimento e água dos animais. Após 13 semanas todos os animais foram eutanasiados através da administração de uma overdose da associação de xilazina com ketamina e realizada a sua necrópsia completa. Os órgãos dos animais foram recolhidos, registadas as suas massas e fixados em formaldeído para posterior análise histopatológica, avaliação imunohistoquímica do marcador de proliferação celular Ki-67; foi também recolhido sangue por punção cardíaca para a realização do ensaio do cometa. Durante o ensaio experimental nenhum dos animais manifestou sinais clínicos de doença. Um murganho do grupo controlo morreu durante o ensaio devido a fatores externos, sendo excluído da experiência. Observámos um maior consumo de água e comida no grupo induzido e alimentado com 20% de FFF. O grupo induzido e alimentado com 50% FFF apresentou o maior aumento de massa corporal ao longo do ensaio. A média da massa relativa do colon do grupo induzido e alimentado com 20% FFF foi superior aos restantes grupos e estatisticamente significativa (p<0,05 ). Os parâmetros recolhidos durante o ensaio relativamente ao bem-estar geral dos animais revelaram não existir diferenças estatisticamente significativas entre grupos. Relativamente à análise histopatológica, 50% (n=6) dos animais do grupo induzido + 20% FFF desenvolveram displasia do epitélio intestinal, 16,67% (n=2) apresentaram adenocarcinomas no reto, sendo o grupo a apresentar mais lesões quando comparado aos restantes. Nos três grupos induzidos com AOM/DSS foi observada inflamação leve a moderada. Relativamente à expressão do marcador de proliferação celular Ki-67, verificou-se uma maior marcação no grupo induzido + 20%FFF. O ensaio do cometa não revelou diferenças estatisticamente significativas entre grupos. Em conclusão, tendo em conta os resultados obtidos, o feijão frade não revela efeitos negativos ou positivos aos animais induzidos quimicamente com AOM/DSS. Novos testes devem ser realizados usando diferentes doses do composto carcinogénico, tempo de exposição ao agente inflamatório, momento do sacrifício e/ou concentração do feijão-frade usado.
Colorectal cancer (CCR) is a worldwide common disease. Most cases are spontaneous and difficult to prevent based on environmental factors and dietary habits. The demonstration of the association between individual health status improvement and cancer mortality rates reduction with adherence to certain diets, such as the Mediterranean diet, the population is encouraged to follow a dietary pattern similar to Mediterranean diet, but not all foods have equal activities. With this research, we intended to evaluate the effects of black-eye-beans supplementation in an induced CCR mouse model by azoxymethane (AOM) followed by dextran-sodium sulfate (DSS) on physiological parameters, histological parameters and genotoxicity. Forty-eight (n=48) female FVB/n mice (Mus musculus) with 5-6 months-old were randomly divided into four groups: control group (n=9), induced group (n=13), induced group + diet containing 20% (m/m) of black-eye-bean flour (BEBF) (n=12) and induced group + diet containing 50% (m/m) of BEBF (n=14). A single intraperitoneal AOM injection was administered (7.5 mg/kg). One week later, animals were exposed to 1.5% of DSS in drinking water for 7 days in three cycles with 7-days interval. All animals had ad libitum access to their diets for 13 weeks. Animals were daily observed to check their general health status. Animals’ body temperature, weight, food and water consumption were also recorded. At the end of the 13 weeks period all animals were humanely sacrificed with an overdose (xylazine + ketamine association) and a complete necropsy was performed. The organs were collected and fixed in formaldehyde to perform histopathological analysis. The genotoxicity of black-eye-beans was determined by the comet assay. None of these animals showed any clinical signs of disease during the experimental period; however, one mouse from control group died during the experiment due to external factors and was excluded from the experience. Water and food consumption registered a slightly higher consumption by the induced group + 20% BEBF when compared to the other groups. The induced group + 50% BEBF showed a higher body mass at the end. The relative colon mass was higher in induced group + 20% BEBF compared to the other groups (p<0,05). The parameters collected during the test for the general welfare of the animals revealed no statistically significant differences between groups. Regarding histopathological analysis, 50% (n=6) of the animals of the induced group + 20% BEBF developed dysplasia and 16.67% (n=2) had rectal adenocarcinoma, being the group presenting more lesions when compared to the other groups. Mild to moderate inflammation was observed in the three groups induced with AOM/DSS. Regarding the expression of the Ki-67 marker, a higher proliferation rate was observed in the induced group + 20% BEBF. The comet assay did not reveal statistically significant differences between groups. In conclusion, considering the results obtained, the black-eye-beans supplementation did not reveal a negative or positive effect to the animals with induced colorectal cancer. Further tests should be performed using different doses of the carcinogenic compound, exposure time to the inflammatory agent, time of sacrifice and/or black-eye-bean concentration.
Colorectal cancer (CCR) is a worldwide common disease. Most cases are spontaneous and difficult to prevent based on environmental factors and dietary habits. The demonstration of the association between individual health status improvement and cancer mortality rates reduction with adherence to certain diets, such as the Mediterranean diet, the population is encouraged to follow a dietary pattern similar to Mediterranean diet, but not all foods have equal activities. With this research, we intended to evaluate the effects of black-eye-beans supplementation in an induced CCR mouse model by azoxymethane (AOM) followed by dextran-sodium sulfate (DSS) on physiological parameters, histological parameters and genotoxicity. Forty-eight (n=48) female FVB/n mice (Mus musculus) with 5-6 months-old were randomly divided into four groups: control group (n=9), induced group (n=13), induced group + diet containing 20% (m/m) of black-eye-bean flour (BEBF) (n=12) and induced group + diet containing 50% (m/m) of BEBF (n=14). A single intraperitoneal AOM injection was administered (7.5 mg/kg). One week later, animals were exposed to 1.5% of DSS in drinking water for 7 days in three cycles with 7-days interval. All animals had ad libitum access to their diets for 13 weeks. Animals were daily observed to check their general health status. Animals’ body temperature, weight, food and water consumption were also recorded. At the end of the 13 weeks period all animals were humanely sacrificed with an overdose (xylazine + ketamine association) and a complete necropsy was performed. The organs were collected and fixed in formaldehyde to perform histopathological analysis. The genotoxicity of black-eye-beans was determined by the comet assay. None of these animals showed any clinical signs of disease during the experimental period; however, one mouse from control group died during the experiment due to external factors and was excluded from the experience. Water and food consumption registered a slightly higher consumption by the induced group + 20% BEBF when compared to the other groups. The induced group + 50% BEBF showed a higher body mass at the end. The relative colon mass was higher in induced group + 20% BEBF compared to the other groups (p<0,05). The parameters collected during the test for the general welfare of the animals revealed no statistically significant differences between groups. Regarding histopathological analysis, 50% (n=6) of the animals of the induced group + 20% BEBF developed dysplasia and 16.67% (n=2) had rectal adenocarcinoma, being the group presenting more lesions when compared to the other groups. Mild to moderate inflammation was observed in the three groups induced with AOM/DSS. Regarding the expression of the Ki-67 marker, a higher proliferation rate was observed in the induced group + 20% BEBF. The comet assay did not reveal statistically significant differences between groups. In conclusion, considering the results obtained, the black-eye-beans supplementation did not reveal a negative or positive effect to the animals with induced colorectal cancer. Further tests should be performed using different doses of the carcinogenic compound, exposure time to the inflammatory agent, time of sacrifice and/or black-eye-bean concentration.
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Cancro colorretal , murganho