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Revista chilena de anatomía

Print version ISSN 0716-9868

Rev. chil. anat. vol.19 n.2 Temuco Aug. 2001 



* Valéria Maria Gomes Totti; **Ruberval A. Lopes; **Marisa Semprini; ** Miguel A. Sala;
** Maria da Glória C. de Mattos; *** Ii-Sei Watanabe & *José Renán Vieira da Costa.

* Escuela de Farmacia y Odontología de Alfenas (EFOA), Alfenas, MG, Brasil.

 Facultad de Odontología de Ribeirão Preto de la Universidad de São Paulo (FORP-USP),  Ribeirão Preto, SP, Brasil.


Instituto de Ciências Biomédicas de la Universidad de São Paulo (ICB-USP), São Paulo, SP, Brasil.

SUMMARY: The objective of the present study was to characterize morphologically, morphometrically and stereologically the epithelial changes in the floor of the mouth of fetuses from rats that received all-trans-retinoic acid during pregnancy. The results suggest that all-trans-retinoic acid causes the appearance of malformed immature and less developed fetuses.

KEY WORDS: 1. Retinoic acid; 2. Fetus; 3. Rat; 4. Malformations.


Retinoids have specific effects on cell differentiation, particularly of the epithelial cells, and on morphogenesis of a wide variety of cells, tissues and organ s ystems. They also have critical importance during vertebrate development (Mendelson et al., 1992), particularly in craniofacial embryogenesis (Morris, 1993). The most active form of vitamin A, retinoic acid, is also one of the most potent teratogens (Schenefelt, 1972).

In order to obtain more information about the effects of all-trans-retinoic acid on the epithelium of the floor of the fetus mouth, Wistar rats were used because this strain appears to be more sensitive to the toxic effects of retinoic acid on development (Chahoud & Nau, 1989).


  Female Wistar rats (Rattus norvegicus), 100 to 120 days old, kept on a 12 h light - 12 h dark cycle, at 22ºC ±2º and 55% ± 5% relative humidity, were housed in individual cages, with free access to a standard pellet diet (Purina) and tap water. The day of estrous, five females were caged overnight with one male for mating. If sperm was present in the vaginal smear, the next day was defined as the first day of pregnancy (GD 1).

In the 10th day of pregnancy (GD10), a single dose of all-trans-retinoic acid (10 mg/kg body wt in sesame oil) was administered to pregnant rats by gavage. Control animals received a similar volume of saline.

Rats were killed by anesthetic ether inhalation on GD 20. The numbers of implantation sites, viable fetuses and resorptions were recorded, and the fetuses were weighed and examined for external malformations. The fetuses, placentas and umbilical cords were fixed in a solution of 85 ml 80% alcohol, 10 ml formalin and 5 ml acetic acid, for 24 h. The material were embedded in paraffin, serially sectioned at 6 µm and stained with hematoxylin and eosin.

For the karyometric study, the histological sections were analised with a light microscope (Jenamed, Jena) with an attached drawing tube (Jena). The contours of the cellular nuclei were drawn in paper with a final magnification of 1000 x, having the care of considering only the elliptic images. In the obtained drawings the larger and smaller diameters were measured.  

The following parameters were estimated in the nuclei of the epithelial cells of the floor of the mouth in control and treated rat fetuses, according to the methods described by Sala et al. (1994): mean diameter, ratio larger diameter to shorter diameter (D/d), perimeter, area, volume, ratio volume to area, coefficient of shape, contour index and eccentricity.  

For the stereological study, the obtained sections of each experimental group were analyzed at the light microscope, with a final magnification of 400x. In the basal and spinous layers of the floor of the mouth epithelium, they were determined the mean cell volume, the number density, surface density (Sala et al.,1992) and the thickness of the cellular layer (Weibel, 1969), employing the grid of Merz (1968).

Statistical comparison between the results obtained for the experimental and control groups was performed by the non-parametric test of Mann-Whitney (Conover, 1999).


Mean fetal weight was 4.07 g for the control group and 1.29 g for the treated group (p< 0.01). Placental weight was 472.0 mg for the control group and 350.2 mg for the treated group (p< 0.01). The mean umbilical cord length was 1.94 cm for the control group and 1.56 cm for the treated group (p<0.01).

Histopathological analysis revealed that the lining epithelium of the floor of the mouth was thinner in the treated group fetuses, constituted by the basal and spinous layers. The number of both cells and mitosis were highly increased. The lamina propria showed fine interlacing collagen fibers with areas of edema, whereas the blood vessels were dilated and congested. (Figs. 1 and 2).

Fig. 1. Histological picture of control fetus floor of the mouth mucosa. Hematoxylin and eosin (900 x).

 Fig. 2. Histological picture of treated fetus floor of the mouth mucosa, showing thinner lining epithelium with both smaller cells and nuclei. Hematoxylin and eosin (900 x).

Karyometric study showed that the larger diameter, smaller diameter, mean diameter, volume, area, perimeter and V/A ratio of nuclei of both basal cell and spinous cell were significantly smaller in the treated group fetus than in the control group fetus (Table I). The nuclear shape was significantly altered mainly in the basal cell, where nuclei appeared more elongated in the treated group fetus than in the control one, as demonstrated by the D/d ratio, eccentricity and coefficient of shape (Table I). In the spinous cells, only the nuclear eccentricity showed to be significantly larger in the treated group animals than in the control group ones (Table I).

Stereological study showed that cytoplasm and cell volumes of both basal cell and spinous cell were significantly smaller in the treated group fetus than in the control group fetus (Table II). Nucleus/cytoplasm ratio, on the other hand, was significantly larger in the treated group than in the control one, in both cellular layers (Table II). Number density of cells was significantly larger in both layer of the treated group than in the control one, whereas the thickness of the basal layer was larger and the thickness of the spinous layer was smaller in the treated group than in the control group (Table II).

The surface density of the epithelium and the numerical density of the epithelial cells were larger, whereas the epithelium thickness of the floor of the mouth was smaller in the treated group fetuses (Table II).


Tretinoin and isotretinoin are known to be teratogenic, inducing retinoic acid embryopathy in man, characterized by defects of the external hearing meatus, hypoplasia of both cerebellum and thymus, and cardiovascular anomalies. However, the dose used in the present study only affected fetal growth, without any gross malformation.

The fetus was significantly lighter in the treated group than in the control group. However, the most important part of fetal development occurs between the 15th and the 21st day of pregnancy, and in this study retinoic acid started acting on the 10th day of pregnancy. Thus, delayed fetal growth may be the expression of cell damage induced before and manifesting during the phase of fetal growth.

Two other factors, placental weight and umbilical cord length, should be taken into consideration when delayed growth or gain of weight occurs. Placenta weight was 472.0 mg for the control fetus and 350.20 mg for the treated group fetus. Drugs and other chemical agents rapidly cross the placental barrier, reaching the developing fetus, although the mechanisms involved in this process of transfer have not been defined. It has been suggested that foreign substances cross the placenta by simple diffusion and the surface of the organ is known to have the characteristics of a lipoid barrier. Other factors such as molecular weight and extent of ionization are of secondary importance (Moya & Thorndike, 1962; Villee, 1965). In addition, the placenta undergoes important changes during pregnancy and these changes are accompanied by modifications in placental permeability (Huggett & Hammond, 1952).

The changes observed in this study support the findings of Kraft et al. (1987, 1989, 1991, 1993), Eckhoff et al. (1989), Gunning et al.(1993), Ward & Kay (1995) and Tzimas et al. (1995, 1997), which demonstrated placental transfer of retinoic acid. Kraft et al. (1991) showed that all-trans-retinoic acid metabolite has concentrations one to three times higher in the placenta than in the maternal plasma, and Tzimas et al. (1994) reported that the 13-cis-retinoic acid isomer reaches the embryo to a much lesser extent that the trans isomer. Johansson et al. (1997) proposed that a number of intra- and extracellular vitamin A-binding proteins exist in embryos and that the amniotic sac mediates its transfer to the embryo or fetus during pregnancy.

Small placentas have reduced blood flow, which results in significant fetal hypoxia and thus might cause intrauterine growth retardation (Emmanouilides et al., 1972). Umbilical cord length is influenced by at least two factors: the incidence of fetal movement and the available intrauterine space (Miller et al., 1981; Moessinger, 1983). If intrauterine space is reduced or if fetal movement is blocked, umbilical cord stretching will be decreased, with the consequent reduction in length. Shorter umbilical cords have been observed in babies with structural limb defects that limit fetal movements (Miller et al.). After injecting curare in pregnant rodents in order to increase amniotic sac pressure and suppress fetal movements, Moessinger observed shorter umbilical cords. Our results showed a shorter umbilical cord in treated animals, indicating limitation of fetal movements. Walker et al. (1968) showed that human umbilical cord growth is small during the last trimester of pregnancy since the cord length of preterm infants was similar to that of term infants. These investigators also observed no correlation between cord length and maternal age or pre-eclampsia, or with mode of infant presentation, sex, weight or body length. Malpas (1964) found no correlation between human umbilical cord length and placental weight or newborn weight.

Since Cohlan (1953) discovered the teratogenic effects of vitamin A, several investigators tried to determine the mechanisms that would explain the malformations and structural alterations of fetuses with no soft tissue malformations. Even though this is a controversial subject, vitamin A probably induces morphogenic defects in embryos or fetuses by:

1. Changing the migration of cells of the cephalic neural crest (Nanda, 1971; Morris, 1975; New, 1978 and Wei et al., 1999);

2. Inhibiting cell proliferation of the ectomesenchyme (Giroud et al., 1961; Marin-Padilla, 1966; Myers et al., 1967; Nanda and New, 1978);

3. Retarding or inhibiting cell differentiation (Kalter & Deuschle, 1966 and Lewis et al., 1978);

4. Inducing multiple effects on cell activity (Walker & Crain ; Kochhar & Johnson, 1965; Lorente & Miller, 1978; or

5. Acting cytotoxically, as demonstrated by ultrastructural studies (Morris, 1973; Theodosis & Fraser, 1978).

In the present study, the mucosa of the floor of the mouth in fetuses treated with retinoic acid showed little differentiation and had the aspect of a more embryonic tissue. Retinol and retinoic acid are known to play a significant role in embryogenesis (Ong & Chytil, 1976) and their presence is necessary to guarantee the differentiation of the various embryonic processes. Thus, vitamin A may have two effects: 1) retarding growth and changing tissue organization, particularly the cephalic mesenchyme (Marin-Padilla; Morris, 1973; Theodosis & Fraser), with a possible reduction of both epithelial (Langman & Welch, 1967) and ectomesenchymal development (Marin-Padilla; Hassell et al., 1978),  and 2) Inhibiting cell differentiation (Kalter & Warkany, 1961; Kalter & Deuschle; Lewis et al.; Hassell et al., 1978) or modifying cell differentiation (Wolf & Varandiani, 1960; Pelc & Fell, 1960 and Langman & Welch, 1967).

The epithelial cells of the floor of the mouth in treated animals showed larger nuclear diameter and higher number density than in the controls. Several authors demonstrated that retinoic acid has the properties of an endogenous morphogen and that embryo tissues contain nuclear receptors specific for retinoic acid (Satre et al., 1992; Grummer et al., 1994, Taylor et al., 1995 andSharma & Kim, 1995).


On the basis of the results obtained for pregnant rats submitted to the effects of oral administration of 10 mg/kg body weight of retinoic acid (tretinoin) on the 10th day of pregnancy, we may conclude that the drug caused a significant decrease in fetal and placental weight and umbilical cord length. With respect to the epithelium of the mouth floor, the drug caused a reduction of the basal and spinous layer thickness, with smaller and more numerous cells, and a disarrangement of basal and spinous cells

In summary, retinoic acid acts on embryogenesis, causing changes in development, with the birth of smaller fetuses, and retards the differentiation of the fetal mouth floor mucosa. In addition, it causes placentas of smaller size and shorter umbilical cords. In general, the fetuses showed a more immature appearance.


This work was supported by grants from the CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico and UNIFRAN.

RESUMEN: El objetivo del presente trabajo fue caracterizar morfológica, morfométrica y estereológicamente las alteraciones del piso de la boca de fetos de rata, provocadas por el ácido retinoico cuando es administrado en el 10º día de preñez.

Los resultados obtenidos sugieren que el ácido retinoico actúa en la embriogénesis provocando alteraciones del desarrollo embrionario.

PALABRAS CLAVE: 1. Ácido retinoico; 2. Feto; 3. Rata; 4. Malformaciones.

Dirección para correspondencia:

Prof. Dr. Ruverbal Armando Lopes
Profesor Titular de Patología
Facultad de Odontología de Ribeirão Preto
Universidade de São Paulo
CEP 14040-904
Ribeirão Preto - SP

Recibido : 15-05-2001
Aceptado: 06-07-2001


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