Int. J. Morphol, 25(4): 689-694,2007.

Effects of Aspartame on Fetal Kidney: A Morphometric and Stereological Study

Efectos del Aspartame en el Riñon Fetal: Estudios Morfométrico y Estereológico

Marielza R. Ismael Martins & Reinaldo Azoubel

Post Graduate Program in Health Sciences - School of Medicine at São Jose do Rio Preto, SP, Brazil.

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SUMMARY: The objective of this study was the evaluation of aspartame effects on morphometric alterations of the glomerulus, proximal and distal convoluted tubules and collecting ducts of the rat fetal kidney during organogénesis. Fifteen pregnant rats averaging 24 g body weight, were divided into 3 groups (n=5 each) of controls, rats treated with aspartame exposed to room temperature and rats treated with aspartame heated to 40°C. Animals were given 14mg / kg aspartame by the intragastric route on the 9^th, 10^th, and 11^th days of pregnancy. Karyometric and stereological techniques estimated morphological changes. A significant decrease of fetal body weight was observed in the group given aspartame kept at room temperature, compared to controls. Karyometry permitted the estimation of the significant nuclear variations observed in the cells of the glomerulus, proximal and distal convoluted tubules and collecting ducts of aspartame-treated rat fetuses. Stereolgical parameters showed statistically significantly increased cell volume and decreased numerical cell density in fetal kidneys of rats treated with aspartame heated to 40° compared to controls. These results indicate that the use of aspartame leads to alterations in all renal structures studied, suggesting this product's nephrotoxicity.

KEYWORDS: Aspartame; Kidney; Morphometry.

RESUMEN: El objetivo de este estudio fue evaluar las alteraciones morfológicas del aspartame en glomérulos, túbulos contorneados distal y proximal y ductos colectores, en ríñones de ratas, durante la organogénesis. Se utilizaron ratas preñadas con un peso promedio de 24g, las que fueron separadas en 3 grupos (n=5 cada uno): un grupo control, otro grupo con ratas tratadas con aspartame y expuestas a temperatura ambiente y un grupo de ratas tratadas con aspartame y mantenidas a 40° C. A los animales se les administró 14 mg/kg de aspartame vía intragástrica en las semanas 9, 10 y 11 de preñez. Con técnicas cariométricas y estereológicas se estimaron los cambios morfológicos. Una disminución significativa del peso fetal se observó en el grupo que recibió aspartame y mantenida a temperatura ambiente, comparado con los controles. La cariometría permitió la estimación de significativas variaciones nucleares observadas en las células de los glomérulos, túbulos contorneados distal y proximal y ductos colectores, en los fetos de las ratas tratadas con aspartame. Los parámetros estereológicos mostraron un incremento estadísticamente significativo del volumen celular y una disminución en la densidad de número de las células, en ríñones fetales de ratas tratadas con aspartame y mantenidas a 40° C comparado con los controles. Estos resultados indican que el uso del aspartame lleva consigo alteraciones en todas las estructuras renales estudiadas, lo que sugiere la nefrotoxicidad del producto.

PALABRAS CLAVE: Aspartame; Riñon; Morfometría.

INTRODUCTION

Aspartame is the methylester of N-alpha-aspartyl-L-phenylalanine, a polypeptide widely employed as an artificial, intense synthetic sweetener, having none or insignificant caloric values and a sweeting power higher than that of sucrose. Its use in pharmaceutical products and in nearly 6000 food products, has been approved; among its consumers are those that replace natural foods by "light" ones (AFSSA. 2002).

Aspartame was discovered in 1965; it took 17 years to be approved for consumption by the United States Food and Drug Administration (FDA) in 1981 (Stengik, 1987), and 29 years for its approval by the European Union, in 1994 (ABIAD, 1994). These periods were marked by controversy about its innocuousness that led to requests for toxicological research that still persists at the present time (AIC, 2005).

Several studies on laboratory animals have been made to verify aspartame's toxicity. Recently, a very large experiment confirmed that it is is a multipotential carcinogenic agent when given at a daily dose of 20 mg/kg body weight, an amount well below the acceptable daily dose of 40mg/kg body weight (Sofritti et al., 2004).

Since 1976, several studies have discussed the need of comments on two aspects of aspartame metabolism: production of phenylalanine and relationship to phenylacetonuria, and the appearance of methanol leading to the formation of methanolic acid, and extremely toxic compound to our organism at already relatively low amounts (Liesovouri &Heikki,1991; Davoli,1986; Goerss etal.,2000).

The increased market of dietary products and the development of new synthetic sweetening compounds have not been sufficiently explored. Thus, to verify the risks and benefits of a substance present in our day-by-day use like aspartame, leads us to worry about the actions of its metabolites (aspartic acid, phenylalanine and methanol) to our organism (Boehm & Bada, 1984; Ishi, 1981).

Although renal excretion is the major process for the removal of toxic substances, renal participation in the assimilation of many substances including aspartame, has not been considered.

Bearing in mind their indiscriminate utilization, the understanding of how certain peptides are metabolized by the kidney, may fill in up to now existing gaps in the understanding of their morphometric effects on this organ (CardelloefaZ.,2001).

The aim of the present study was to evaluate the effect of aspartame on the fetal kidney of rats, by observing through karyometry and stereology, structural changes in the glomeruius, proximal and distal convoluted tubules and collecting duct.

MATERIAL AND METHOD

Fifteen albino femaleWistar rats averaging 220-g bodyweight were kept in individual cages and fed a commercial ration and water ad libitum. The study protocol was approved by the Ethics Committee for animal experimentation of the Faculty of Medicine of São José do Rio Preto, SP, Brazil. Animals were mated overnight with males at the proportion of 1:4; in the morning their vaginal smears were examined for presence of spermatozoids, and if so, were exposed on the 9^th, 10^th and 11^th day of pregnancy i.e., during the period of organogénesis (68), to a 14 mg/kg body weight dose of aspartame.

On the 20^th day of pregnancy, the animals were sacrificed, fetuses collected, immediately immersed in Alfac solution, fixed for 5 days, and weighted on a precision balance. To obtain permanent histological preparations, samples from 30% of the treated and control aleatorily chosen offspring, were obtained. Fetal kidneys were dissected and 6 |j.m slices cut in semi-serial way, and stabilized with hematoxilin-eosin. Morphometry utilized karyometric and stereological evaluations of fetal kidney glomeruli, convoluted proximal and distal tubules and collector ducts.

Glomerular volumes were estimated using an optical Hund 11500 Wetzlar microscope (Helmut Hund Gmbh, Germany), with a40x objective, and a Leitz Wetzlar camera lucida, yielding a final 490-fold magnification. Glomerule images were projected on white sulfite paper and their contours drawn with a black, N° 2 pencil. Twenty structure drawings were obtained from each animal, totalizing 150 for each group.

Karyometry was employed to evaluate nuclear shape and volume as well as their relationships, by averaging the largest and smallest diameters of the epithelial cell nuclei of the proximal and distal convoluted tubules and the renal duct collectors. Slices were analyzed under the above-described microscope with a 100 x objective provided with a Leitz Wetzlar camera lucida, at a final 1240 fold magnification. The contours of the 50 images obtained from each structure were drawn as described above, care being taken to annotate only elliptical images. A millimeter ruler was used to obtain the largest (D) and the smallest (d) axis diameters of the images. These diameters, as well as mean diameters, their ratios, areas, perimeters, volume/area relationships, eccentricity, contour indexes, shape factor and coefficients were evaluated. For stereology, the Merz (Merz, 1968) grid was utilized: slices were focussed under an optical microscope with a 100x objective, and a camera lucida. Images obtained were drawn on the grid. The aim was to obtain cytoplasm volumes, cell volumes, nuclear/cytoplasm relations and numerical density, by counting points remaining superposed to the nuclei and cytoplasm of these cells and on sinusoids of the fetal regions of treated groups, heat-treated groups and controls respectively, totaling the utilization of 20 different fields.

A computer program in Basic Advanced language, developed by Professors Geraldo Maia Campos and Miguel Angel Sala, from the Stomatology Department of the Odontology School of Ribeirao Preto, USP, was utilized. Comparisons between groups were performed by Mood's test for medians, Kruskal-Wallis test, and the Mann-Whitney test, when significant (P<0.05) differences had been found.

RESULTS

Results for initial and final body weight parameters.

Results for the quantitative parameters placenta weight and umbilical - cord length for control rat fetuses and those treated with aspartame are shown in Table II.

Glomeruius. It is to be noted that the kidney volume of the kidney of animals given room temperature-treated aspartame and animals given 40°C-treated, dissolved preparation, were significantly higher than that of the control group (Table III).

Proximal convoluted tubules. Results demonstrated that there was no statistically significant difference between the size of the lesser diameter and the ratio greatest/ smallest diameters, and that there was no modification of the cell shape factor corresponding to the eccentricity, contour index and shape coefficient (Table IV).

Distal convoluted tubules showed increased nuclear diameters, implying an increased perimeter, area and volume of these nuclei in fetuses from both groups of treated rats. The shape of these nuclei (eccentricity, shape coefficient and contour index), presented a significant alterations in animals given room temperature-treated aspartame (Table V).

Collecting ducts revealed significant variations of parameters like the greater diameter, medium diameter, area, perimeter and volume/area ratio in the group given 40° entreated, dissolved aspartame. In both treated groups, volumes increased significantly, but there was no significant variation of nuclear shapes compared to controls (Table VI).

Stereology. The data shown on Table VII, present the increase of cell and cytoplasm volumes of aspartame-treated groups. On cell quantification, a significant reduction of the proximal convoluted as well as disappearance of many of these structures at the brush border of 40°C-treated, diluted aspartame rat fetuses. At the distal convoluted tubules, cell nuclei also were found in lesser numbers.

The glomeruli of the treated groups also showed all their stereological parameters to be altered, with larger, but a reduced number of epithelial cells. Nevertheless, the collection ducts did not present statistical differences regarding their numerical density.

DISCUSSION

The present study showed that daily orogastric administration of 14mg/kg of aspartame on the 9^th, 10^th and 11^th day of pregnancy, led to alterations in the development of renal structures.

Limited studies had been performed and published by others, who administered aspartame from the 7^th to the 14^th day of pregnancy, an important period for organogénesis in the rat (Sturtevant, 1985; Holder, 1989; Lennon et a/.,1980).

In the present experiments, we observed morphological alterations that revealed that aspartame crossed the placenta causing a reduction of fetal weight, following the administration, when kept at room temperature or when diluted and heated to 40°C .This result agrees with those by others that administered 20mg aspartame /kg body weight daily from the 10^th to the 14^th day of pregnancy and detected delayed fetal development. Our study suggests that administration of aspartame on the 9^th, 10^th and 11^th day of pregnancy delays fetal growth as expressed by cell damage during this period.

These findings demonstrate the usefulness of the animal model employed for the study of the effects of aspartame on the developing kidney by karyometry and stereology quantification, leading to the finding that there occurs an alteration of kidney structures during pregnancy.

Our observations that aspartame toxicity became more pronounced following its dilution and heating to 40° C, also agreed with those of Tsang (1985) who also used the experimental model. Their study reports that any form of heating of aspartame, rapidly causes the formation of diketopiperazine (DKP) and free phenylalanine. Significant amounts of DKP are also formed when aspartame is stored in solution at room temperature, but heating considerably hastens this process.

It appears therefore that further work aimed at the elucidation of the mechanisms by which this artificial sweetener determines nuclear and cell alterations in the kidney, is needed.

Based on our results it is to be concluded that karyometry and stereology analyses of groups of fetal rats, suggests nephrotoxicity to the glomerulus, proximal and distal convoluted tubules and to a lesser degree, the collecting ducts, of aspartame kept at room temperature or diluted and heated to 40° C.

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Correspondence to:

Prof. Dr. Reinaldo Azoubel
Faculdade de Medicina de São José do Rio Preto
Av. Brigadeiro Faria Lima 5416
CEP 15090-000.
São José do Rio Preto - SP - BRASIL

Email: guilherme@famerp.br

Received: 23-04-2007 Accepted: 07-08-2007