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Revista chilena de anatomía

Print version ISSN 0716-9868

Rev. chil. anat. vol.16 n.2 Temuco  1998

http://dx.doi.org/10.4067/S0716-98681998000200002 

EFFECTS OF LOW-DOSE EXPOSURE OF RAT FETUSES PANCREAS TO METHYLMERCURY. MORPHOMETRIC AND STEREOLOGIC STUDIES

EFECTOS DE LA EXPOSICION A BAJAS DOSIS DE METILMERCURIO SOBRE EL PANCREAS DE FETOS DE RATAS. ESTUDIOS MORFOMETRICO Y ESTEREOLOGICO

                * Glória E. F. Mendes
                * Reinaldo Azoubel
                ** Ruberval A. Lopes
                ** Miguel A. Sala
                ** Suzie A. de Lacerda
                ** Amadeu R. Silva Júnior

MENDES, G. E. F.; AZOUBEL, R.; LOPES, R. A.; SALA, M. A.; de LACERDA, S. A. & SILVA Jr., A. R. Effects of low-dose exposure of rat fetuses pancreas to methylmercury. A morphometric and stereologic studies. Rev. Chil. Anat., 16(2):177-183, 1998.

SUMMARY: Methylmercury chloride (20 mg/kg body weight), when administered orally to Wistar rats on day 10 of pregnancy, originated smaller fetuses and placentas. Death and resorptions were present and no gross malformations were seen. Histologically, the larger pancreatic acini of treated fetus showed irregular shape with larger pyknotic nuclei. The nuclei varied in size and shape. The lumen was small. In the faintly eosinophilic cytoplasm of the cells less numerous secretory granules were present. The ducts were disorganized and presented larger nuclei. The pancreatic islets were immature and less organized. The data were analyzed with morphometric and stereologic techniques.

KEY WORDS: 1. Methylmercury; 2. Rat; 3. Fetus; 4. Pancreas; 5. Morphometry; 6. Stereology.

INTRODUCTION

Methylmercury can readily cross the placenta, accumulating in the fetus, where it can deleteriously affect the offspring (KOOS & LONGO, 1960). Prenatal methylmercury chloride (MMC) administration was seen to result in high frequency of fetal malformations. The congenital anomalies included cleft palate, skeletal deformities, and hydronephrosis (HARRIS et al., 1972; CURLE et al., 1987). Other effects of MMC observed in rat fetuses are spina bifida, syndactyly, retarded growth, subcutaneous edema, ventral wall defects, and pericardial distention (MELKONIAN & BAKER, 1988).

The objective of the present study was to investigate the changes in the glandular epithelium of the pancreas occurring in fetuses from pregnant rats that had received methylmercury during pregnancy.

MATERIAL AND METHOD

Ten sexually mature Wistar rats were kept in one room at a constant temperature and under a controlled 12-hour light cycle with free access to water and commercial rat food. Males were placed with females overnight and the females examined the following morning for the presence of sperm plugs in a vaginal smear. The day of finding plugs was designed as day 1 of pregnancy.

MMC was administered by using an intragastric tube, on day 10 at dose level of 20 mg/kg of body weight. Control animals received a corresponding volume of isotonic saline. On day 20 of gestation, the pregnant rats were anesthetized and laparotomized. Fetuses were placed in a fixative solution of 85 ml 80% alcohol, 10 ml formalin and 5 ml acetic acid, for 24 hours. The fetuses were dissected free of the placenta, umbilical cord and all fetal membranes and then were weighed.

Serial 6 µm sections were taken through the transverse plane from middle abdomen at the level of the pancreas and stained with haematoxylin and eosin.

The nuclear measurements of acinar and duct cells of the pancreas of 10 fetuses were calculated according SALA et al. (1994). The longest axis (D) and the shortest axis (d) were measured in the drawing of each nucleus in order to estimate the following nuclear parameters:

1. Geometric mean axis: M = (D · d)1/2
2. Ratio of longest to shortest axis: D/d
3. Perimeter: P = (p /2) · [(3/2) · (D + d) - M]
4. Area: A = (p /4) · M2
7. Volume: V = (p /6) · M3
8. Ratio of volume to area: V/A
9. Shape factor: F = 4 · p · A/P2
10.Contour index: I = P/ (A)1/2
11. Eccentricity: E = (D + d)1/2· (D - d)1/2/ D

Stereological analysis was performed with a curvilinear test system of MERZ (1968).

The measurement of volume density of acini and ducts was performed by the point-counting method. Thus, the volume density of a component can be estimated by the fraction of test points lying on the structure.

The surface density of acini and ducts were estimated according to TOMKEIEFF (1945):

Sv = 2 · (I / Lt)

where I is the number of intersection points of the test lines with the surface of the structure and Lt is the total length of the test lines.

The mean diameter of pancreatic acini and ducts was estimated according to SALA et al. (1980):

D = Pe · z / I

where Pe is the number of test points lying on the structure, z is the test line length and I is the number of intersection points of the test line with the surface of the structure.

The mean thickness of both acini and ducts epithelial layer was estimated after SALA et al (1981):

T = (Pv · z / 2 · Iv) · [1 - (Pl / Pv)1/2 ]

where Pv and Pl are the number of points lying on the structure and the lumen, respectively; and Iv is the number of intersection points of the test line (of length = z) with the surface of the structure.

Comparison of the results for the experimental and the control groups was made by the nonparametric Mann-Whitney test.

RESULTS

The mean fetal body weight was 4.29 g for the control group and 2.35 g for the methylmercury-treated group (p < 0.01). The placenta weight was 554.88 mg for the control and 455.18 mg for the treated-group (p < 0.01). Death and resorptions were present.

Histologically, the pancreatic parenchyma differs markedly from the control fetus when observed at 20th day of pregnancy. The normal aspect of the pancreas is showed in Figs. 1 and 3. The larger acini of methylmercury-treated fetus showed irregular shape with larger pyknotic nuclei. The nuclei varied in size and shape. The lumen was small. In the faintly eosinophilic cytoplasm of the cell less numerous secretory granules were present (Fig. 2). The ducts were disorganized and presented larger nuclei. The pancreatic islets were immature and less organized (Fig. 4).

Fig. 1. Histological picture of the pancreas of control rat fetus. Note the pyramidal cells which compose the acini; the cytoplasm full of acidophilic zymogen granules and the spherical nuclei. Haematoxylin and eosin (1000 x).

Fig. 2. Pancreas of methylmercury-treated rat fetus. Note larger acini of irregular shape with pyknotic nuclei of higher volume and the cytoplasm, faintly eosinophilic with less numerous secretory granules. Haematoxylin and eosin (1000 x).

Fig. 3. Pancreatic islets of control fetus. Note the islet cells separated from the tissue of the exocrine pancreas. Haematoxylin and eosin (1000 x).

Fig. 4. Pancreatic islets of treated rat fetus. Note the immature and less organized islet cells. Haematoxylin and eosin (1000 x).

Tables 1 and 2 show the principal mean values of morphometric parameters of the pancreatic structures.

The acini and ducts nuclear longest, shortest and geometric mean axis, perimeter, area, volume, ratio V/A and shape factor were smaller in the control group. The contour index and eccentricity were higher in the acini nucleus of the control group. Statistical analysis showed significant differences between the control group and the treated group.

The mean acini diameter and wall thickness were higher in methylmercury-treated fetus. The surface density was smaller in treated group.

DISCUSSION

In the present paper, epithelial alterations were observed in the pancreas of fetuses where mothers were submitted to methylmercury during pregnancy. These alterations were demonstrated on data obtained by karyometry (larger acini and ducts cell nuclear volumes), and stereology (increased acinar mean diameter and wall thickness, decreased duct surface density). All these findings demonstrate a delayed differentiation of the analyzed tissue in the treated animal.
 
Table 1. Mean values of karyometric and stereologic parameters of acini of pancreas for control and methylmercury-treated Wistar rat fetuses. Mann-Whitney test.
 

Parameter Control Treated Ucalc  P[U]
Karyometry        
Longest axis (µm) 8.00 8.80 0 0,004
Shortest axis (µm) 5.91 6.93 0 0,004
Geometric mean axix (µm) 6.87 7.80 0 0,004
Ratio D/d 1.35 1.27 1 0,008
Perimeter (um) 22.05 24.81 0 0,004
Area (µm2) 37.16 48.00 0 0,004
Volume (µm3) 170.54 250.60 0 0,004
Ratio V/A 4.58 5.21 0 0,004
Shape factor 0.96 0.98 1.5 0,012
Contour index 3.60 3.58 2 0.016
Eccentricity 0.67 0.61 1 0.008
Stereology        
Mean diameter (µm) 55.50 65.20 3 0.028
Wall thickness (µm) 23.10 28.44 0 0.004
Surface density (mm3/mm3) 437.70 401.94 11 0.421
Volume density (%) 57.34 64.90 5 0.075
Volume density connective tissue (%) 27.86 23.41 9 0.275

U calc = Calculated Mann-Whitney U value
P[U] = Probability of occurrence of the calculated U value

Table 2. Mean values of karyometric and stereologic parameters of ducts of pancreas for control and methylmercury-treated Wistar rat fetuses. Mann-Whitney test.
 
Parameter Control Treated Ucalc  P[U]
Karyometry        
Longest axis (µm) 7.78 8.55 0 0,004
Shortest axis (µm) 5.67 6.27 1 0,008
Geometric mean axix (µm) 6.64 7.32 0 0,004
Ratio D/d 1.37 1.36 11 0,421
Perimeter (um) 21.26 23.43 0 0,004
Area (µm2) 34.65 42.23 0 0,004
Volume (µm3) 153.53 207.04 0 0,004
Ratio V/A 4.43 4.88 0 0,004
Shape factor 0.96 0.96 12 0,500
Contour index 3.61 3.61 12 0.500
Eccentricity 0.68 0.68 12 0.500
Stereology        
Mean diameter (µm) 75.32 82.54 10 0.345
Wall thickness (µm) 28.19 23.47 5 0.075
Surface density (mm3/mm3) 79.50 54.54 0 0.004
Volume density (%) 11.49 27.86 0 0.004

U calc = Calculated Mann-Whitney U value
P[U] = Probability of occurrence of the calculated U value

The mechanism by which methylmercury disturbs mammalian development and produces alterations in the pancreas is unknown. Methylmercury can pass the placental barrier and cause fatal damage which may not be evident at birth (TAKAHASHI et al., 1971; KHERA, 1972). According to MANSOUR et al. (1974), methylmercury freely crosses the placenta, reaching the fetus in larger amounts than mercury nitrate, regardless of the day of pregnancy or the time after injection. This fact is probably related to the property of liposoluble substances if rapidly crossing the cell membranes, with methylmercury being soluble in lipids. The placental weight reduction observed in the present study was also reported by HOLT & WEBB (1986), and small placentas have impaired circulation with reduced blood flow to the fetus and thereby impair nutrition, which results in fetuses with smaller body weight.

In this paper, it was observed a significantly lower body weight of fetuses from animals intoxicated with methylmercury. Administration of methylmercury on the 9th day of pregnancy at doses of 10, 15 and 20 mg/kg caused a dose-dependent reduction in fetal weight (CURLE et al., 1987). This decrease in fetal weight was probably due to the reduced mitotic index of treated fetuses (INOUYE & MURAKAMI, 1975; SKALKO et al., 1971; CURLE et al., 1983). Methylmercury inhibits DNA, RNA and protein synthesis (SLOTKIN et al., 1985; SARAFIAN & VERITY, 1986) and ruptures the microtubules of the mitotic apparatus (MIURA et al., 1978). Although the dose used in the present study was not teratogenic, the drug may accumulate in the fetus, as demonstrated by INOUYE et al. (1986) and may probably have a toxic effect.

Mercury has long been known to carry proteins that form mercaptide bonds with sulphydryl groups and other lateral chains of amniotic proteins, resulting in denaturation and deactivation of protein molecules (RAMEL, 1969; CURLE et al., 1983; SAGER et al., 1984). The affinity of mercury for biopolymer anions is a basic mechanism of mercury toxicity resulting in many reactions between cell protein and mercury. Poisoned enzyme systems result in a reduced energy supply and lead to entropy (BRUBAKER et al., 1971, 1973). Protein synthesis is inhibited, resulting in a reduced rate of cell growth and development (THRASHER et al., 1972; BRUBAKER et al., 1973).

Increased free radical formation may lead to greater DNA damage. Intrinsic enzyme (superoxide dismutase) protect against free radical formation and this protection is reduce when this enzyme system is inhibited by mercury. A high rate of free radical formation may be an important and sudden mechanism of cell toxicity induced by mercury (SINGH et al., 1984).

The alterations provoked by methylmercury ingestion in pregnant rats were quite evident in our results (treated fetuses) concerning the pancreatic epithelium.

It should be pointed out that, eve though the present data clearly show histological alterations highly similar to those reported in the literature, they also suggest that further studies should be carried out using more sophisticated methods to study the ultrastructural alterations caused by the action of methylmercury.

MENDES, G. E. F.; AZOUBEL, R.; LOPES, R. A.; SALA, M. A.; de LACERDA, S. A. & SILVA Jr., A. R. Efectos de la exposición a bajas dosis de metilmercurio sobre el páncreas de fetos de ratas. Estudios morfométrico y estereológico. Rev. Chil. Anat., 16(2):177-183, 1998.

RESUMEN: El cloruro de metilmercurio (20 mg/kg de peso corporal), cuando fue administrado oralmente a ratas Wistar en el día 10 de la preñez, dio origen a fetos y placentas de menor peso. Hubo muerte y reabsorción fetal, a pesar de no existir malformaciones macroscópicas. Histológicamente, los acinos pancreáticos, aumentados de tamaño, mostraron forma irregular con grandes núcleos picnóticos en los animales tratados. Los núcleos variaron en tamaño y forma. El lumen de los acinos era menor. En el citoplasma, débilmente eosinófilo, se observaron escasos gránulos de secreción. Los ductos eran desorganizados y presentaron núcleos mayores. Los islotes pancreáticos eran inmaduros y menos organizados. Las alteraciones morfológicas fueron analizadas mediante técnicas morfométricas y estereológicas.

PALABRAS CLAVE: 1. Metilmercurio; 2. Rata; 3. Feto; 4. Páncreas; 5. Morfometría; 6. Estereología

This work was supported by the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) - Proc. No 350.535/90-2.
* School of Medicine of São José do Rio Preto, SP, Brazil.
** School of Dentistry of Ribeirão Preto, University of São Paulo, SP, Brazil.

Dirección para correspondencia:
Prof. Dr. Ruberval A. Lopes
Departamento de Estomatologia (Patologia)
Faculdade de Odontologia de Ribeirão Preto - Universidade de São Paulo
Av. do Café, s/n
14.040-904 Ribeirão Preto, SP,
BRASIL


Recibido : 23-02-1998
Aceptado: 28-06-1998

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