Efeito do ácido fólico em três modelos experimentais de anomalias anorretais em ratos
Data
2015-08-27
Autores
Faria, Danilo Jose Fiorindo 
Orientadores
Martins, Jose Luiz Martins
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: As anomalias anorretais (AAR) são defeitos congênitos da porção terminal intestinal com incidência de 1:5000 nascidos vivos. Diferentes drogas com efeito teratogênico têm sido utilizadas experimentalmente na tentativa de mimetizar as AAR, dentre elas a Etilenotiouréia (ETU), a Adriamicina (ADR) e os precursores e análogos da vitamina A (VA). Objetivo: Avaliar três diferentes modelos experimentais de indução de AAR em ratos e qual o efeito do ácido fólico (AF) neles. Métodos: Foram utilizadas 25 ratas Wistar, com prenhes induzida. As ratas foram distribuídas aleatoriamente em 6 grupos: Grupo A 1 - ETU; Grupo A 2 - ADR; Grupo A 3 - VA; Grupo B 1 - AF + ETU; Grupo B 2 - AF + ADR; Grupo B 3 - AF + VA. As ratas dos grupos B receberam, diariamente, 2 semanas antes e durante toda a prenhes, ácido fólico, 50mg/kg, por gavagem. As ratas dos Grupos 1, A e B receberam Etilenotiouréia, 125 mg/Kg à 1%, por gavagem, no D11. As ratas dos Grupos 2, A e B receberam Adriamicina, 6mg/kg, intraperitoneal, no D8. As ratas dos Grupos 3, A e B receberam o Ácido Retinóico, 100mg/kg no D9, por gavagem. No D21, as ratas foram submetidas à cesariana. Os filhotes foram examinados para determinação da presença de AAR. Foram analisadas a espessura do epitélio estratificado pavimentoso anal (EEPA) e a espessura do epitélio intestinal (EI) de cada filhote. Nível de significância p menor ou igual 0.05. Resultados: Obtivemos 269 filhotes. O número de filhotes; número de AAR; média (Delta%) ± DP são respectivamente: ETU - 49;30;65%±24%. AF+ETU - 52;01;02%±03%. ADR - 41;29;65%±37%. AF+ADR - 40;04;16%±36%. VA -40;00;00%±00%. AF+VA - 47;00;00%±00%. Houve diferenças estatisticamente significantes entre ETU e VA, e entre ADR e VA (p=0,025), mas não houve diferenças estatisticamente significantes entre ETU e ADR. Não houve diferenças estatisticamente significantes com uso de AF sobre VA (p> 0,05). O ácido fólico reduziu significativamente o número de AAR com ETU (p=0,025) e reduziu o número de AAR com ADR (p=0,05). As médias de espessura do EEPA ± DP e do EI ± DP foram respectivamente: ETU - 27,75±0,56; 18,88±0,93. AF+ETU - 28,88±0,61; 21,11±0,16. ADR - 25,98±0,74; 19,48±1,68. AF+ADR - 24,74±0,91; 24,80±0,81. VA - 55,08±1,21; 41,85±0,48. AF+VA - 54,12±0,32; 43,87±0,34. A espessura do EEPA e EI estão diminuídas com ADR e ETU (p<0,001), e houve aumento da espessura do EI quando utilizado AF (p<0,005). Discussão: Obtivemos 65% de AAR induzidas por ETU, condizente com a literatura, assim como anomalias de membros e coluna vertebral. O AF foi capaz de reduzir significativamente a formação de AAR (p<0,005). Encontramos 65% de AAR induzidas por ADR no D8, resultados pouco diferentes da literatura, por serem modelos diferentes. Mostramos que foi possível induzir AAR com apenas uma dose de ADR. O AF reduziu o número de AAR (p=0,05), exercendo certo efeito protetor nestes modelos, já que a incidência de AAR caiu de 65% para 16%. Não foi possível reproduzir os achados esperados quando induzidos com VA. Uma das possibilidades foi o uso de Ácido Retinóico, e não o ATRA. A espessura do EEPA e EI estão diminuídas com uso de ETU e ADR, mas não com uso de VA. Conclusões: As drogas ADR e ETU induzem AAR nos modelos propostos, não sendo possível dizer qual melhor modelo. O ácido fólico reduz significativamente o número de AAR quando induzidas por ETU, e reduz o número de anomalias quando induzidas por ADR.
Background: Anorectal malformations (ARMs) are congenital defects of hindgut development. ARMs have an incidence of 1/5000 live births. Different teratogenic drugs have been used to induce ARMs. The most currently used are ethylenethiourea (ETU), Adriamycin (ADR) and metabolites and precursors of vitamin A (VA). Purpose: The aim of this study was to evaluate three different experimental models to induce ARMs and determine the effect of folic acid (FA) in those models. Methods: 25 female Wistar rats were divided randomly in 6 groups. Group A 1 - ETU; Group A 2 - ADR; Group A 3 - VA; Group B 1-FA + ETU; Group B 2- FA + ADR; Group B 3 - FA + VA. Dams from group B received daily, since 2 weeks before the pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from groups 1, A and B, received 1% ETU (125mk/kg) by gavage on gestational day (GD) 11. Dams from groups 2, A and B, received ADR (6mk/kg) by intraperitoneal injection on GD8. Rats from groups 3, A and B, received Retinoic Acid (100mk/kg) by gavage on GD9. Their embryos were harvested by cesarean section on GD21 and were examined looking for ARMs. The thickness of anal stratified squamous epithelium (ASSE) and intestinal epithelium (IE) were analyzed. P values less than or equal to 0.05 were considered significant. Results: 269 embryos were harvested. The number of ARMs; number of embryos; statistic mean ± SD were determined to be, respectively: ETU - 49;30;65%±24%. FA+ETU - 52;01;02%±03%. ADR - 41;29;65%±37%. FA+ADR - 40;04;16%±36%. VA - 40;00;00%±00%. FA+VA - 47;00;00%±00%. Presence of AMRs in ETU and ADR groups were significantly larger than VA group (p=0.025). There were no significant differences between ETU and ADR groups. There were no significant differences between FA+VA and VA groups (p> 0.05). AMRs were significantly lower in FA+ETU group than in ETU group (p=0.025). AMRs were lower in FA+ADR group than in ADR group (p=0.05). The mean thickness (um) of ASSE± SD and IE ± SD were measured, respectively: ETU - 27.75±0.56; 18.88±0.93. FA+ETU - 28.88±0.61; 21.11±0.16. ADR - 25.98±0.74; 19.48±1.68. FA+ADR - 24.74±0.91; 24.80±0.81. VA - 55.08±1.21; 41.85±0.48. FA+VA - 54.12±0.32; 43.87±0.34. The thickness of ASSE and IE were significantly lower in ADR and ETU groups (p<0.001). The thickness of IE were significantly enlarged when FA was given (p<0.005). Discussion: ARMs induced by ETU were observed in 65%, similar to other papers. Limbs and spine anomalies were similar too. FA was able to, significantly, protect against ARMs ETU-induced. ARMs induced by ADR were observed in 65%, slightly different from other papers that used another experimental model. A single dose of ADR on D8 was able to induce ARMs. FA was relatively able to protect against ARMs ADR-induced, by reducing ARMs incidence from 65% to 16%. We were not able to reproduce ARMs induced by VA models. One of reasons could be the use of Retinoic Acid instead of ATRA. The thickness of ASSE and IE were thinner in ADR and ETU groups than in VA groups. Conclusion: ETU and ADR induced ARMs in these models, but were not possible to define which the best was. Folic acid significantly reduces the number of ARMs ETU-induced and marginally reduces the number of ARMs ADR-induced.
Background: Anorectal malformations (ARMs) are congenital defects of hindgut development. ARMs have an incidence of 1/5000 live births. Different teratogenic drugs have been used to induce ARMs. The most currently used are ethylenethiourea (ETU), Adriamycin (ADR) and metabolites and precursors of vitamin A (VA). Purpose: The aim of this study was to evaluate three different experimental models to induce ARMs and determine the effect of folic acid (FA) in those models. Methods: 25 female Wistar rats were divided randomly in 6 groups. Group A 1 - ETU; Group A 2 - ADR; Group A 3 - VA; Group B 1-FA + ETU; Group B 2- FA + ADR; Group B 3 - FA + VA. Dams from group B received daily, since 2 weeks before the pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from groups 1, A and B, received 1% ETU (125mk/kg) by gavage on gestational day (GD) 11. Dams from groups 2, A and B, received ADR (6mk/kg) by intraperitoneal injection on GD8. Rats from groups 3, A and B, received Retinoic Acid (100mk/kg) by gavage on GD9. Their embryos were harvested by cesarean section on GD21 and were examined looking for ARMs. The thickness of anal stratified squamous epithelium (ASSE) and intestinal epithelium (IE) were analyzed. P values less than or equal to 0.05 were considered significant. Results: 269 embryos were harvested. The number of ARMs; number of embryos; statistic mean ± SD were determined to be, respectively: ETU - 49;30;65%±24%. FA+ETU - 52;01;02%±03%. ADR - 41;29;65%±37%. FA+ADR - 40;04;16%±36%. VA - 40;00;00%±00%. FA+VA - 47;00;00%±00%. Presence of AMRs in ETU and ADR groups were significantly larger than VA group (p=0.025). There were no significant differences between ETU and ADR groups. There were no significant differences between FA+VA and VA groups (p> 0.05). AMRs were significantly lower in FA+ETU group than in ETU group (p=0.025). AMRs were lower in FA+ADR group than in ADR group (p=0.05). The mean thickness (um) of ASSE± SD and IE ± SD were measured, respectively: ETU - 27.75±0.56; 18.88±0.93. FA+ETU - 28.88±0.61; 21.11±0.16. ADR - 25.98±0.74; 19.48±1.68. FA+ADR - 24.74±0.91; 24.80±0.81. VA - 55.08±1.21; 41.85±0.48. FA+VA - 54.12±0.32; 43.87±0.34. The thickness of ASSE and IE were significantly lower in ADR and ETU groups (p<0.001). The thickness of IE were significantly enlarged when FA was given (p<0.005). Discussion: ARMs induced by ETU were observed in 65%, similar to other papers. Limbs and spine anomalies were similar too. FA was able to, significantly, protect against ARMs ETU-induced. ARMs induced by ADR were observed in 65%, slightly different from other papers that used another experimental model. A single dose of ADR on D8 was able to induce ARMs. FA was relatively able to protect against ARMs ADR-induced, by reducing ARMs incidence from 65% to 16%. We were not able to reproduce ARMs induced by VA models. One of reasons could be the use of Retinoic Acid instead of ATRA. The thickness of ASSE and IE were thinner in ADR and ETU groups than in VA groups. Conclusion: ETU and ADR induced ARMs in these models, but were not possible to define which the best was. Folic acid significantly reduces the number of ARMs ETU-induced and marginally reduces the number of ARMs ADR-induced.
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Citação
FARIA, Danilo Jose Fiorindo. Efeito do ácido fólico em três modelos experimentais de anomalias anorretais em ratos. 2015. 85 f. Dissertação (Mestrado em Ciência Cirúrgica Interdisciplinar) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.