Effect of folic acid in a modified experimental model of anorectal malformations adriamycin-induced in rats.

PURPOSE: To determine the effect of a single dose of adriamycin (ADR) to induce anorectal malformations (ARMs) and determine the effect of folic acid (FA) in this model. METHODS: Ten female Wistar rats were divided randomly in two groups. Group A - ADR; Group B - FA+ADR. Dams from group B received daily, since two weeks before the pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from both groups received ADR (6mk/kg) by intraperitoneal injection on gestational day (GD) 8. Their fetuses 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≤0.05*. RESULTS: 81 fetuses were harvested. The number of fetuses; number of ARMs; mean (∆%) (± SD) were determined to be, respectively: ADR - 41[29;65%(±37%)] versus FA+ADR - 40[04;16%(±36%)] (p=0.05). AMRs were significantly lower in FA+ADR group than in ADR group (p=0.05). The thickness (µm) of ASSE (± SD) and IE (± SD) were measured, respectively: ADR - [25.98(±0.74) and 19.48(±1.68)] versus FA+ADR - [24.74(±0.91) and 24.80(±0.81)] (p<0.005). The thickness of IE was significantly enlarged when FA was given (p<0.005). CONCLUSIONS: Single dose of adriamycin on D8 was able to induce anorectal malformations. Folic acid reduces the number and enlarged the IE of ARMs ADR-induced.

Effect of folic acid in a modified experimental model of anorectal malformations adriamycin-induced in rats

PURPOSE: To determine the effect of a single dose of adriamycin (ADR) to induce anorectal malformations (ARMs) and determine the effect of folic acid (FA) in this model. METHODS: Ten female Wistar rats were divided randomly in two groups. Group A - ADR; Group B - FA+ADR. Dams from group B received daily, since two weeks before the pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from both groups received ADR (6mk/kg) by intraperitoneal injection on gestational day (GD) 8. Their fetuses 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≤0.05*. RESULTS: 81 fetuses were harvested. The number of fetuses; number of ARMs; mean (∆%) (± SD) were determined to be, respectively: ADR - 41[29;65%(±37%)] versus FA+ADR - 40[04;16%(±36%)] (p=0.05). AMRs were significantly lower in FA+ADR group than in ADR group (p=0.05). The thickness (µm) of ASSE (± SD) and IE (± SD) were measured, respectively: ADR - [25.98(±0.74) and 19.48(±1.68)] versus FA+ADR - [24.74(±0.91) and 24.80(±0.81)] (p<0.005). The thickness of IE was significantly enlarged when FA was given (p<0.005). CONCLUSIONS: Single dose of adriamycin on D8 was able to induce anorectal malformations. Folic acid reduces the number and enlarged the IE of ARMs ADR-induced.

Genetic research and structural dysplasia assessment of anorectal malformations in neonatal male rats induced by di(n-butyl) phthalate.

This study was the first to investigate the genetic abnormalities and structural dysplasia of anorectal malformations (ARMs) in male rats induced by di(n-butyl) phthalate (DBP). DBP was administered to timed-pregnant rats to establish the ARM rat model. The incidence of ARMs in male offspring was 39.5%. In neonatal period, decreased body weight and anogenital distance were observed. The general image and histological analysis of male offspring confirmed the presence of ARMs. Anatomical examination of the ARM male rats revealed the dysplasia in solid organs (heart-lung, liver, spleen, and kidney). The decreases of serum testosterone concentration and androgen receptor expression in terminal rectum were indicative of the antiandrogenic effects of DBP. Moreover, significant decreased mRNA expressions of these androgen-related genes such as sonic hedgehog, Gli2, Gli3, bone morphogenetic protein 4, Wnt5a, Hoxa13, Hoxd13, fibroblast growth factor 10, and fibroblast growth factor receptor 2 were found in terminal rectum of the ARM male pubs. These results demonstrated that development of ARM rats was impaired by maternal exposure to DBP. The antiandrogenic effects of DBP disturbing the androgen-related signaling networks might play an important role in the occurrence of ARMs.

Reduced Fgf10/Fgfr2 and androgen receptor (AR) in anorectal malformations male rats induced by di-n-butyl phthalate (DBP): A study on the local and systemic toxicology of DBP.

Previous study have demonstrated that not only the anorectal development but also the general conditions of anorectal malformations (ARMs) male rats are severely affected by di-n-butyl phthalate (DBP) maternal exposure. However, the mechanisms underlying DBP-induced congenital defects remain elusive. Reportedly, Fgf10/Fgfr2 and androgen receptor (AR) are pivotal for the development of multiple organs. In this study, we therefore investigated the expression of Fgf10/Fgfr2 together with AR in the terminal rectum and multiple organs of ARM male rats induced by in utero exposure to DBP. DBP was administered to pregnant rats to establish the model and the incidence of ARMs in male offspring was 39.5%. On postnatal day(PND)1, the gross photograph and histopathological staining confirmed the abnormal manifestations in these organs of newborn ARMs. Decreased anogenital distance, body weight and serum testosterone level were observed in ARM male offspring. The reduced expression of Fgf10/Fgfr2 mRNA and protein was seen in terminal rectum and kidney, spleen, liver, heart in ARM male rats, whereas the reduced expression of AR was only observed in the kidney and terminal rectum. Our findings suggest the potential involvement of altered Fgf10/Fgfr2 signaling and AR in pathogenesis of local and systemic development defects in ARMs male rats induce by DBP.

Is it possible folic acid reduce anorectal malformations ethylenethiourea induced in rats?

PURPOSE: To investigate the effect of folic acid (FA) in an experimental model of anorectal malformations (ARMs) ethylenethiourea (ETU) induced. METHODS: Eight female Wistar rats were divided randomly in two groups. Group A - ETU; Group B - FA+ETU; Dams from group B received daily, since two weeks before pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from groups A and B, received 1% ETU (125 mk/kg) by gavage on gestational day (GD) 11. Their fetuses 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 < 0.05*. RESULTS: One hundred and one embryos were harvested. The number of embryos; number of ARMs; mean statistical % (± SD) were determined to be, respectively: ETU - 49 [30;65% (± 24%)] versus FA+ETU - 52 [1;02% (± 3%)] (p = 0.025). AMRs were significantly lower in FA+ETU group than in ETU group (p = 0.025). The thickness (µm) of ASSE (± SD) and IE (± SD) were measured, respectively: ETU - [27.75 (± 0.56) and 18.88 (± 0.93)] versus FA+ETU - [28.88 (± 0.61) and 21.11 (± 0.16)] (p = 0.001). The thickness of IE was significantly enlarged when FA was given (p=0.001). CONCLUSION: Folic acid reduces the number and enlarged the IE of ARMs ETU-induced.

Is it possible folic acid reduce anorectal malformations ethylenethiourea induced in rats?

PURPOSE: To investigate the effect of folic acid (FA) in an experimental model of anorectal malformations (ARMs) ethylenethiourea (ETU) induced.METHODS:Eight female Wistar rats were divided randomly in two groups. Group A - ETU; Group B - FA+ETU; Dams from group B received daily, since two weeks before pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from groups A and B, received 1% ETU (125mk/kg) by gavage on gestational day (GD) 11. Their fetuses 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<0.05*.RESULTS:One hundred and one embryos were harvested. The number of embryos; number of ARMs; mean statistical % (± SD) were determined to be, respectively: ETU - 49 [30;65% (±24%)] versus FA+ETU - 52 [1;02% (±3%)] (p=0.025). AMRs were significantly lower in FA+ETU group than in ETU group (p=0.025). The thickness (µm) of ASSE (± SD) and IE (± SD) were measured, respectively: ETU - [27.75 (±0.56) and 18.88 (±0.93)] versus FA+ETU - [28.88 (±0.61) and 21.11 (±0.16)] (p=0.001). The thickness of IE was significantly enlarged when FA was given (p=0.001).CONCLUSION:Folic acid reduces the number and enlarged the IE of ARMs ETU-induced.

Spatiotemporal expression of BMP7 in the development of anorectal malformations in fetal rats.

The aim of this study was to determine the expression patterns of bone morphogenetic protein 7 (BMP7) during anorectal development in normal rat embryos and in embryos with anorectal malformations (ARM), and to investigate the possible role of BMP7 in the pathogenesis of ARM. ARM was induced by treating rat embryos with ethylenethiourea on the 10th gestational day (GD10). Embryos were harvested by Cesarean delivery and the spatiotemporal expression of BMP7 was evaluated in normal (n=168) and ARM embryos (n=171) from GD13 to GD16 using immunohistochemistry staining and western blot analysis. Immunohistochemical staining in normal embryos revealed that BMP7 was abundantly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13, and BMP7-immunopositive cells were extensively detected in the URS, hindgut, and cloacal membrane by GD14. Increased positive tissue staining was noted on the fused tissue of the URS and the thin anal membrane on GD15. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negatively or only faintly immunostained for BMP7. BMP7 protein expression showed time-dependent changes in the developing hindgut according to western blotting, and reached a peak on GD15 during anus formation. BMP7 expression levels from GD14 to GD15 were significantly lower in the ARM group compared with the normal group (P<0.05). Spatiotemporal expression of BMP7 was disrupted in ARM embryos during anorectal morphogenesis from GD13 to GD16. These results suggest that downregulation of BMP7 at the time of cloacal separation into the primitive rectum and UGS might be related to the development of ARM.

Spatiotemporal expression of Cdx4 in the developing anorectum of rat embryos with ethylenethiourea-induced anorectal malformations.

PURPOSE: The aim of this study was to determine the expression of Cdx4 (caudal-type homeobox gene-4) during anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx4 in ARM pathogenesis. MATERIALS AND METHODS: ARM was induced by ETU on the 10th gestational day (GD10) in rat embryos. Cesarean deliveries were then performed to harvest the embryos. Spatiotemporal expression of Cdx4 was evaluated in normal rat embryos (n = 354) and ARM embryos (n = 378) from GD13 to GD16. RESULTS: Immunohistochemical staining and immunofluorescence revealed that, in normal embryos, Cdx4 expression was extensively detected on the epithelium of the cloaca on GD13. On GD14, the Cdx4-positive cells were intensively detected on the hindgut. On GD15, the anal membrane was constantly immunoreactive to Cdx4. On GD16, Cdx4-labeled cells were observed on the epithelium of the anus. In the ARM embryos, the epithelium of the cloaca, urorectal septum (URS) and anorectum was negative or faint for Cdx4. In the normal embryo group, Cdx4 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16 on Western blot and real-time reverse transcription plus polymerase chain reaction. Once the URS divided the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx4 expression began to decrease. In addition, the expression level of Cdx4 in the ARM group from GD13 to GD15 was significantly lower than that in the normal group (p < 0.05). CONCLUSIONS: In ARM embryos, an imbalance in the spatiotemporal expression of Cdx4 was noted during anorectal morphogenesis from GD13 to GD16. This suggests that ETU may cause downregulation of Cdx4 expression. Downregulation of Cdx4 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.

Spatiotemporal expression of Wnt5a during the development of the striated muscle complex in rats with anorectal malformations.

UNLABELLED: Fecal incontinence and constipation after procedures for anorectal malformations (ARMs) are closely related to the maldevelopment of the striated muscle complex (SMC). Previous studies have demonstrated that myogenic regulatory factors (MRFs) play a significant role in muscle development. Wnt signal pathway is extremely important for MRFs regulation. This study was designed to investigate the spatiotemporal expression pattern of Wnt5a in SMC in ARMs rat embryos. MATERIALS AND METHODS: Anorectal malformation embryos were induced by ethylene thiourea on embryonic day 10 (E10). Expression levels of protein and mRNA of Wnt5a were confirmed by immunohistochemistry staining, western blot and quantitative real-time PCR (qRT-PCR) between normal rat embryos and embryos with ARMs. RESULTS: Immunostaining revealed that, on embryonic day 17 (E17), the Wnt5a protein was initially expressed in the SMC in normal embryos. With the growth of pregnancy, the positive staining cells gradually increased. The same time-dependent changes of Wnt5a protein were detected in ARMs embryos. Besides, immunostaining showed that Wnt5a had a significant increase in normal embryos compared with ARMs embryos. Similarly, in Western blot and qRT-PCR, the higher expression of Wnt5a protein and mRNA were remarkable in normal embryos during the SMC development, relatively. CONCLUSION: Our study demonstrated that the downregulation of Wnt5a at the time of SMC development might partly be related to the dysplasia of SMC in ARMs.

Expression of Wnt5a during development of anorectal malformations in a rat model of prenatal exposure to di(n-butyl) phthalate.

Mounting evidence has indicated the crucial role of Wnt5a in the embryonic development including guts. However, the Wnt5a involvement in the process of anorectal malformations (ARMs) remains unclear. In this study, we examined the expression of Wnt5a during ARMs development in the offspring of di(n-butyl) phthalate (DBP)-treated pregnant rats. During the neonatal period, Wnt5a expression was evaluated in the terminal rectum of ARM offspring, non-ARM littermates and controls. Using real-time polymerase chain reaction (real-time PCR), western-blot analysis and immunohistochemistry approaches, we found a significant decrease of Wnt5a expression in DBP-induced ARMs rats. Collectively, our results demonstrate the aberrant expression of Wnt5a during anorectal development, which suggests that Wnt5a might be involved in DBP-induced ARMs.

Temporal and spatial expression of caudal-type homeobox gene-2 during hindgut development in rat embryos with ethylenethiourea-induced anorectal malformations.

The main aim of this study was to determine Cdx2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos, and Cesarean deliveries were performed to harvest the embryos. The temporal and spatial expression of Cdx2 was evaluated in normal rat embryos (n = 303) and ARM embryos (n = 321) from GD13 to GD16. Immunohistochemical staining revealed that, in normal embryos, Cdx2 was mainly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13. On GD14, Cdx2-immunopositive cells were extensively detected on the URS, hindgut, and cloacal membrane. On GD15, increased immunopositive tissue staining on the anal membrane was evident. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negative or faintly immunostaining for Cdx2. Analyses by Western blot and real-time reverse transcription plus the polymerase chain reaction revealed that, in the normal group, Cdx2 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16. Upon the URS division of the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx2 expression began to decrease. Moreover, the Cdx2 expression level in the ARM group from GD13 to GD14 was significantly lower than that in the normal group (P < 0.05). In ARM embryos, an imbalance in the spatiotemporal expression of Cdx2 was noted during anorectal morphogenesis from GD13 to GD16. Downregulation of Cdx2 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.

Abnormal neural crest innervation in Sox10-Venus mice with all-trans retinoic acid-induced anorectal malformations.

BACKGROUND/PURPOSE: Despite technical advances in the surgical/medical care of anorectal malformation (ARM), persistent unsatisfactory postoperative bowel habit has been attributed to histopathologic abnormalities of the distal rectum/pouch (DRP) and hypoplasia of anal sphincter muscles (ASM). We used Sox10-Venus mice with ARM induced by all-trans retinoic acid (ATRA) to investigate neural crest cell (NCC) innervation in the DRP and ASM. METHOD: Pregnant Sox10-Venus mice were administered single doses of 50, 70, or 100 mg/kg of ATRA on embryonic day 8.5 (E8.5) then sacrificed on either E16.5 or E19.5. Bowel specimens comprising the anorectum were examined using fluorescence microscopy without immunohistochemical staining (FMIS). Anti-PGP9.5 was used to delineate ganglion cells and anti-SMA for smooth muscles. RESULTS: The appropriate dose of ATRA for inducing ARM was 50 mg/kg. Under FMIS, all ARM embryos (n = 5; all high type; 3 male:2 female) had less NCC innervation with thick Venus-positive nerve fibers in the DRP compared with normal embryos (n = 8); there was abnormal NCC innervation in the DRP and absent ASM in ARM mice. CONCLUSION: We are the first to delineate abnormal enteric nervous system innervation in the DRP of ARM mice without using immunohistochemical staining techniques thus allowing specimens to be examined without any distortion.

Maternal asthma medication use and the risk of selected birth defects.

OBJECTIVES: Approximately 4% to 12% of pregnant women have asthma; few studies have examined the effects of maternal asthma medication use on birth defects. We examined whether maternal asthma medication use during early pregnancy increased the risk of selected birth defects. METHODS: National Birth Defects Prevention Study data for 2853 infants with 1 or more selected birth defects (diaphragmatic hernia, esophageal atresia, small intestinal atresia, anorectal atresia, neural tube defects, omphalocele, or limb deficiencies) and 6726 unaffected control infants delivered from October 1997 through December 2005 were analyzed. Mothers of cases and controls provided telephone interviews of medication use and additional potential risk factors. Exposure was defined as maternal periconceptional (1 month prior through the third month of pregnancy) asthma medication use (bronchodilator or anti-inflammatory). Associations between maternal periconceptional asthma medication use and individual major birth defects were estimated by using adjusted odds ratios (aOR) and 95% confidence intervals (95%CI). RESULTS: No statistically significant associations were observed for maternal periconceptional asthma medication use and most defects studied; however, positive associations were observed between maternal asthma medication use and isolated esophageal atresia (bronchodilator use: aOR = 2.39, 95%CI = 1.23, 4.66), isolated anorectal atresia (anti-inflammatory use: aOR = 2.12, 95%CI = 1.09, 4.12), and omphalocele (bronchodilator and anti-inflammatory use: aOR = 4.13, 95%CI = 1.43, 11.95). CONCLUSIONS: Positive associations were observed for anorectal atresia, esophageal atresia, and omphalocele and maternal periconceptional asthma medication use, but not for other defects studied. It is possible that observed associations may be chance findings or may be a result of maternal asthma severity and related hypoxia rather than medication use.

The expression analysis of Notch-1 and Jagged-2 during the development of the hindgut in rat embryos with ethylenethiourea induced anorectal malformations.

BACKGROUND: This study was designed to investigate the expression of Notch-1 and Jagged-2 in the terminal hindgut in ethylenethiourea (ETU)-exposed rat embryos with anorectal malformations (ARMs) and its potential association with the maldevelopment of the terminal hindgut in ARMs. MATERIAL AND METHODS: ETU-exposed ARMs model was introduced to investigate the expression pattern of Notch-1 and Jagged-2 during the hindgut development using immunohistochemical staining, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: Immunostaining revealed that the expression of Notch-1 and Jagged-2 showed changes in the developing terminal hindgut of ARMs. The expression of Notch-1 and Jagged-2 in the terminal hindgut of ARMs rat embryos decreased at both mRNA level and protein level (P < 0.05) compared with normal tissues. CONCLUSION: This study demonstrated that the expression of Notch-1 and Jagged-2 in ARMs of ETU-exposed rat embryos was remarkably reduced, which implied its potential role in the pathogenesis of the terminal hindgut maldevelopment in ARMs.

The development of satellite cells and their niche in striated muscle complex of anorectal malformations rat embryos.

BACKGROUND: It has been demonstrated that different degrees of pelvic floor muscle (PFM) maldevelop in anorectal malformations (ARMs); yet the development of satellite cells, the myogenic stem cells responsible for muscle growth, repair, and maintenance remains elusive during the embryogenesis of PFM. Striated muscle complex (SMC) is one of the most important components of PFM. The objective of this study was to observe the development pattern of satellite cells and their niche of SMC and investigate its possible role in PFM dysplasia in ARMs. METHODS: Immunohistochemistry, cell culture, transmission electron microscopy (TEM), real-time quantitative PCR, and Western blot were performed to trace the dynamic development pattern of satellite cells during the morphogenesis of PFM in ethylenethiourea (ETU)-induced ARMs rat embryos. RESULTS: In ARMs rat embryos, earlier presentation and higher number of Pax7-expressing cell were observed in SMC. The expression of Pax7 and vimentin were up-regulated, while the expression of myogenin, vWF, and neurofilament were down-regulated. Ultrastructure analysis of SMC was characterized by increased amount of nuclear heterochromatin of satellite cell nuclei, thickened basal lamina, widened gap between satellite cell and myofiber, and disarrangement of muscle fibers. The satellite cells demonstrated abnormal differentiation after they were isolated and cultured in vitro. CONCLUSIONS: Our results suggest that premature origination of satellite cell from myogenic progenitor or precursor may result in the depletion of myogenic precursor and cessation of muscle growth; intrinsic defect in satellite cell structure, and extrinsic impairment of microenvironment compromised the myogenic competence of satellite cell, which might contribute substantially to the hypoplastic SMC in ARMs.

Abnormal innervation patterns in the anorectum of ETU-induced fetal rats with anorectal malformations.

To investigate whether anorectal malformations (ARMs) were associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution of neuronal markers protein gene product (PGP9.5), nitric oxide synthases (NOs), neuromuscular junction markers (synaptophysin, SYP), interstitial cells of Cajal (ICC) marker (c-kit) within the terminal rectum were analyzed by immunohistochemistry and Western blot in rat embryos with ethylenethiourea (ETU) induced ARMs. From Gestational day16 (Gd16) to Gd21, neural crest-derived cells (NCC) migrated from the proximal gut into the terminal colon, colonising it along its entire length, gradually proliferated and differentiated to innervate the distal gut. From Gd19 to Gd21, significant gross-morphological differences of the anorectum of normal (n=90) and ARMs (n=90) embryos were found. Different myenteric plexus (MPs) development of the anorectum suggested that ARMs were associated with a global abnormal innervation patterns in the anorectum in gestational course and might have some postoperative effect.

Study of density of interstitial cells of cajal in the terminal intestine of rats with anorectal malformation.

INTRODUCTION: Intestinal constipation is one of the most commonly occurring complaints in the postoperative period after correction of anorectal malformation (ARM). An abnormal density of interstitial cells of Cajal (ICC) is one potential cause. The objective of this study was to analyze the density of ICC in the terminal intestine of fetuses of rats with anorectal anomaly induced by ethylenethiourea (ETU). MATERIAL AND METHODS: The fetuses were distributed into three groups: Group A--normal fetuses obtained from pregnant female rats that did not receive ETU; Group B--fetuses with no ARM, obtained from pregnant rats that received ETU, and Group C--fetuses with ARM, obtained from pregnant rats that received ETU. ETU was administered on the 11th day of pregnancy at a dose of 125 mg/kg. The fetuses were extracted by means of laparotomy on the 21st day of pregnancy. The terminal intestine of the fetuses was removed and analyzed by immunohistochemistry to evaluate ICC. RESULTS: Statistically significant differences were found between groups A, B and C regarding the density of ICC. Group A presented with the highest density, followed by groups B and C. CONCLUSION: There is a lower density of ICC in the terminal intestine of rats with ARM.

Evaluation of an experimental model for anorectal anomalies induced by ethylenethiourea

PURPOSE: To evaluate an experimental model for anorectal anomalies and their principal associated malformations induced by ethylene thiourea (ETU). METHODS: Rat fetuses were utilized, divided into two groups: experimental group - fetuses from rats that received ETU on the 11th day of gestation at the dose of 125 mg/kg, diluted in distilled water to 1 percent concentration (12.5 ml/kg); and control group - fetuses from rats that received distilled water alone, at a volume of 12.5 ml/kg. On the 21st day of gestation, the animals were sacrificed by hypoxia in a carbon dioxide chamber, followed by laparotomy to remove the fetuses. These were initially examined externally to determine the sex and whether anorectal anomalies and malformations of the vertebral column and tail were present. Then, with the aid of microscopy, the fetuses underwent exploratory laparotomy to characterize the type of anorectal anomaly and investigate urological malformations. RESULTS: None of the fetuses in the control group presented anorectal anomaly, vertebral column malformation or urological structural alterations. In the experimental group, 71 percent presented anorectal anomaly, 80 percent presented vertebral column alterations and 35 percent presented urological alterations. CONCLUSION: The model described was shown to be easy to implement and presented results that allow its use in studying anorectal anomalies and associated malformations.

OBJETIVO: Avaliar o modelo experimental de AAR, induzido pela Etilenotiouréia (ETU), quanto à ocorrência de anomalia anorretal e das principais malformações associadas. MÉTODOS: Foram utilizados fetos de ratos distribuídos em 2 grupos: Grupo experimental - Fetos provenientes de ratas que receberam ETU no décimo primeiro dia de gestação na dose de 125 mg/Kg, diluída em água destilada na concentração de 1 por cento (12,5 ml/Kg) e Grupo controle - Fetos de ratas que receberam somente água destilada num volume de 12,5 ml/Kg. No 21° dia de gestação, os animais foram submetidos à eutanásia por hipóxia em câmara de gás carbônico e laparotomia para retirada dos fetos. Os fetos foram, inicialmente, examinados externamente para determinação do sexo, presença de AAR, de malformações de coluna vertebral e da cauda. A seguir, com o auxílio de microscopia, os fetos foram submetidos a laparotomia exploradora para caracterização do tipo de AAR e investigação de malformações urológicas. RESULTADOS: Nenhum dos fetos do grupo controle apresentou AAR, malformações de coluna vertebral e alterações urológicas estruturais. No grupo experimental, 71 por cento apresentaram anomalia anorretal, 80 por cento apresentaram alterações de coluna vertebral e 35 por cento apresentaram alterações urológicas. CONCLUSÃO: O modelo descrito se mostrou de fácil execução e apresentou resultados que permite o seu emprego no estudo das anomalias anorretais e das malformações associadas.

Evaluation of an experimental model for anorectal anomalies induced by ethylenethiourea.

PURPOSE: To evaluate an experimental model for anorectal anomalies and their principal associated malformations induced by ethylene thiourea (ETU). METHODS: Rat fetuses were utilized, divided into two groups: experimental group - fetuses from rats that received ETU on the 11th day of gestation at the dose of 125 mg/kg, diluted in distilled water to 1% concentration (12.5 ml/kg); and control group - fetuses from rats that received distilled water alone, at a volume of 12.5 ml/kg. On the 21st day of gestation, the animals were sacrificed by hypoxia in a carbon dioxide chamber, followed by laparotomy to remove the fetuses. These were initially examined externally to determine the sex and whether anorectal anomalies and malformations of the vertebral column and tail were present. Then, with the aid of microscopy, the fetuses underwent exploratory laparotomy to characterize the type of anorectal anomaly and investigate urological malformations. RESULTS: None of the fetuses in the control group presented anorectal anomaly, vertebral column malformation or urological structural alterations. In the experimental group, 71% presented anorectal anomaly, 80% presented vertebral column alterations and 35% presented urological alterations. CONCLUSION: The model described was shown to be easy to implement and presented results that allow its use in studying anorectal anomalies and associated malformations.

Reproductive and developmental toxicity screening test of basic rubber accelerator, 1,3-di-o-tolylguanidine, in rats.

Twelve male and female rats per group were exposed to the rubber accelerator 1,3-di-o-tolylguanidine (DTG) by gavage at 0, 8, 20 or 50 mg/kg bw/day. Males were dosed for a total of 49 days beginning 14 days before mating. Females were dosed for a total of 40-49 days beginning 14 days before mating to day 3 of lactation throughout the mating and gestation period. At 50 mg/kg bw/day, deaths were observed in two males and three females. Lowered body weight gain and food consumption were noted in males at 50 mg/kg bw/day and females at 20 and 50 mg/kg bw/day. Mydriasis, decreased locomotor activity, bradypnea, prone position, tremor and/or salivation were observed in males and females at 20 and 50 mg/kg bw/day. No effects of DTG were found on the estrous cyclicity, precoital interval, copulation, fertility and gestational indices, numbers of corpora lutea and implantations, or gestation length. A significant decrease in the number, body weight and viability of offspring and increase in the incidence of fetuses with external malformations were found at 50 mg/kg bw/day. Oligodactyly, anal atresia and tail anomalies were observed. These data suggest that DTG may be teratogenic. The NOAELs of DTG for general and developmental toxicity in rats are 8 and 20 mg/kg bw/day, respectively.