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.


Introduction
Anorectal anomalies include a spectrum of malformations that affect the terminal intestine and other systems such as the urinary, skeletal and cardiovascular systems.The term "imperforate anus", which has become recognized over time, merely reflects the most marked appearance of this entity.Approximately one in every 5,000 live newborns presents anorectal anomaly [1][2][3] .The embryology of the normal terminal intestine, and also the embryology of anorectal anomalies, is controversial and is mainly based on unproven theories.Given the difficulty of obtaining human embryos with anorectal anomaly, studies on their embryogenesis require the use of experimental animal models 4 .In addition to demonstrating morphological aspects of the gastrointestinal tract, animal models also allow study of the intrinsic enteric innervation, the striated musculature and urological and skeletal malformations.The experimental models for anorectal anomalies include those due to mutations, those that occur spontaneously and those induced by drugs.The objective of the present study was to evaluate the experimental model for anorectal anomalies and associated malformations induced by ethylene thiourea (ETU).

Methods
Four pregnant rats of Wistar lineage OUT-B EPM-1 (Rattus norvegicus albinus, Rodentia Mammalia), coming from the central vivarium of the Federal University of São Paulo, Escola Paulista de Medicina, were utilized.During the experiment, the animals received commercial feed and water ad libitum, under constant environmental conditions of temperature and humidity, with 12-hour day and night cycles using artificial light, controlled automatically.After one night of mating, the animals that presented a vaginal smear with the presence of spermatozoids were considered to be potentially fertilized.This was considered to be day zero (D0) of gestation.From this time onwards, the rats were kept in individual cages.On the 11 th day of gestation (D11), three pregnant rats received ethylene thiourea (2imidazolidinethione C 3 H 6 N 2 S 98%, from batch no.24251-089 from Sigma-Aldrich, Brazil), diluted in distilled water at 1% concentration, at a dose of 125 mg/kg (12.5 ml/kg), by intragastric gavage.The fourth animal received distilled water alone, without the addition of ethylene thiourea, at a volume of 12.5 ml/kg.From this time onwards, the animals were defined as two groups: Control group (C): the animal that received distilled water alone, without the addition of ethylene thiourea (ETU); and Experimental group (E): the animals (subgroups E1, E2 and E3) that received ETU.
On the 21 st day of gestation, the animals were sacrificed by means of hypoxia in a carbon dioxide chamber, and underwent laparotomy to remove the fetuses.The fetuses were initially examined externally to determine their sex and whether there were anorectal anomalies and/or malformations of the vertebral column and tail.Following this, with the aid of microscopy, the fetuses underwent exploratory laparotomy to characterize the type of anorectal anomaly and investigate any urological malformations.

Ethics
This experiment received approval from the Research Ethics Committee of the Federal University of São Paulo (UNIFESP-EPM), with protocol no.0406/05.The experiment also received funding from FAPESP, as case no.05/56062-8.

Results
The numbers of fetuses obtained per rat are presented in Table 1.None of the fetuses in the control group presented anorectal anomalies.In the experimental group, nine fetuses from E1 (90%), ten from E2 (91%) and three from E3 (30%) presented anorectal anomalies.The model utilized provoked anorectal anomalies in 71% of all the fetuses studied.The types of anorectal anomaly found were: 14 cloacal malformations (Figure 1), four perineal fistulas and four urethral fistulas (Figures 2-3) (Table 2).None of the fetuses in the control group presented malformations of the vertebral column.However, these were found in 80% of the fetuses in the experimental group (all the fetuses from E1 and E2, and four from E3).The abnormalities found were tail agenesis (Figure 4) or short tail (Table 3).None of the fetuses in the control group presented urological structural alterations.Among the fetuses in the experimental group, 35% presented alterations.Ureteral hydronephrosis (Figure 5) was found in five fetuses, unilateral kidney agenesis in four fetuses, hypoplasia of the left kidney in one fetus, and persistence of the urachus (Figure 6) in one fetus (Table 4).Among the fetuses from subgroup E1, there was an association between anorectal anomaly and malformation of the vertebral column in six fetuses (60%) and an association of anorectal anomaly with both malformation of the vertebral column and urological malformation in three fetuses (30%).Malformation of the column alone was present in one fetus (10%) (Table 5).Among the fetuses in subgroup E2, there was an association between anorectal anomaly and malformation of the vertebral column in four fetuses (36%) and an association of anorectal anomaly with both malformation of the vertebral column and urological malformation in six fetuses (60%).One fetus presented malformation of the column with urological abnormality but without anorectal anomaly (9%) (Table 6).Among the fetuses in subgroup E3, there was an association between anorectal anomaly and malformation of the vertebral column in three fetuses (30%).There was no association of anorectal anomaly with both malformation of the vertebral column and urological malformation.There was malformation of the column alone in one fetus (10%) and urological malformation alone in one fetus (10%) (Table 7).

Discussion
The embryology of anorectal malformations is a controversial subject.It is difficult to study this in human beings because of the scarcity of fetuses with anorectal anomalies.Hence, there is a need for an animal model 4 .The experimental models for anorectal anomalies include those due to mutations, those that occur spontaneously and those induced by drugs.The most classical and oldest model is the one described by Danforth, which is known as "Danforth's short-tail mouse" 5 .This is a natural mutation that presents a series of malformations, and among these is imperforate anus.More recently, a mouse with a mutation involving the "sonic hedgehog" was discovered.This is a signaling molecule that performs a variety of roles in the development of vertebrates, and which exhibits imperforate anus in its phenotype 6 .These animals are difficult to obtain and extremely expensive.The spontaneous occurrence of anorectal malformations is not uncommon among domestic animals, especially among pigs, with incidence of around 0.2%.They present similarities with the malformations in humans, but with a low rate of associated malformations 7 .By crossing affected animals, Hori et al 8  the most frequent type, whereas in humans this is one of the rarest types [1][2][3] .The 80% incidence of abnormalities of the vertebral column was somewhat lower than found by other authors, who have reported tail agenesis or rudimentary tails in 100% of the fetuses, of which 48 to 70% exhibited myeloschisis.We did not find this in our animals 12,14,15 .Urological alterations, which in the human species occur in 13 to 50% of the patients, occurred in 35% of our fetuses.The principal abnormalities found were kidney agenesis and ureteral hydronephrosis, similar to findings among humans.There is no corresponding data in the literature for comparison.The presence of these malformations takes on special interest, since they are the cause of great morbidity and mortality in humans.

Conclusion
The model described was shown to be easy to implement and presented results that allow its utilization for studying anorectal anomalies and their associated malformations.

TABLE 2 -
Classification of the anorectal anomalies found

TABLE 3 -
Malformations of the vertebral column found

TABLE 4 -
Urological structural malformations found

TABLE 5 -
Associations between anomalies for the fetuses in subgroup E1

TABLE 6 -
Associations between anomalies for the fetuses in subgroup E2

TABLE 7 -
Associations between anomalies for the fetuses in subgroup E3 FIGURE 6 -Persistence of the urachus