Efectos del ciclamato de sodio en el rinon fetal de ratas: estudio morfometrico.
RESUMEN: El ciclamato es usado como edulcorante no calorico en muchos alimentos y bebidas y es 30 veces mas dulce que la sacarosa, sin el gusto amargo de la sacarina. Aparecen en su composicion, los productos como ciclamato de sodio, ciclamato de calcio y acido ciclamico. El objetivo del trabajo fue evaluar los efectos del ciclamato de sodio en rinones de fetos de ratas, considerandose las alteraciones morfometricas en glomerulo, tubulos contornados proximal y distal y conducto coletor. Fueron utilizadas 10 ratas adultas (Rattus norvegicus) variedad Wistar, con peso medio de 238 g, 5 ratas para el grupo control y 5 ratas tratadas con ciclamato de sodio. Entre el 10[grados] y 14[grados] dia de la prenez, 5 ratas recibieron una inyeccion diaria intraperitoneal de 60mg/Kg/dia de ciclamato de sodio durante 5 dias. En el 20[grados] dia, los animales fueron sacrificados y los fetos fijados en solucion de Alfac, incluidos en parafina, cortados y tenidos com H-E. El metodo utilizado fue la morfometria por la tecnica cariometrica. Hubo disminucion significativa en los pesos de los fetos y de la placenta en el grupo tratado con ciclamato de sodio (p= 0,004) comparado con el grupo control. En el volumen glomerular y tamano nuclear de las celulas de los tubulos contornados proximal, distal y conducto colector del rinon fetal de las ratas tratadas con ciclamato de sodio, hubo aumento estadisticamente significativo. Los resultados mostraron que el uso del ciclamato de sodio produjo la reduccion del peso de los fetos, placenta y longitud del cordon umbilical. Hubo aumento significativo en el volumen glomerular y en el tamano nuclear de las celulas de los tubulos contornados proximal, distal y conducto colector, sugerente de nefrotoxicidad.
PALABRAS CLAVE: 1. Rinon; 2. Ciclamato de sodio; 3. Morfometria; 4. Estudio experimental.
SUMMARY: The cyclamate is used as artificial non-caloric sweetner in a wide variety of foods and beverages, being 30 times as sweet as sugar without the bitter after-taste of saccharin. It appears in the composition of the products as sodium and calcium cyclamates and cyclamic acid. The purpose of this research was to assess the effect of sodium cyclamate in kidneys of rats fetuses considering morphometric changes of glomerulus, proximal and distal convoluted tubules, and collecting duct. Ten pregnant rat (Rattus norvegicus) weighting an average of 238 g were divided into two groups: 5 in the control group and 5 in treated group with sodium cyclamate. Between the 10th and the 14th pregnancy day, 5 rats received an intraperitoneal daily injection of 60 mg/kg of body weight of sodium cyclamate during 5 days. At the 20th, the animals were sacrificed and the fetuses were fixed in Alfac solution, included in paraffin, sectioned and stained with haematoxyline and eosine. Morphometry by the karyometric technique was the method used. The principal measurements were glomerular volume and cell nuclei size of proximal and distal convoluted tubules and collecting duct. There were a significant decrease in fetal and placentae weight in treated group with sodium cyclamate (p = 0.004) when compared with control group. In the glomerular volume and cell nuclei size of proximal and distal convoluted tubules and collecting duct of kidneys of rats fetuses treated with sodium cyclamate, the increase was statistically significant. The results showed that the use of sodium cyclamate was effective in the reduction of fetal and placentae weight, and lenght of umbilical cords. There was a significant increase in glomerular volume and in cell nuclei size of proximal and distal convoluted tubules and collecting duct, suggesting nephrotoxicity.
KEY WORDS: 1. Kidney; 2. Sodium cyclamate; 3. Morphometry; 4. Experimental study.
The category of cyclamates include salts of the acid N-cyclo-hexyl-sulfamic that are used asa non-caloric artificial sweetener in various foods and beverages and in the pharmaceutical industry. The composition of the product appears in different forms: sodium cyclamate, calcium cyclamate and cyclamic acid (Cattanach, 1976).
In 1969, cyclamate associated with saccharin in a 10:1 proportion was interpreted by the FDA as an inductor of bladder cancer in rats (Price et al., 1970). Based on this study, its use was prohibited in the United States. Despite this, the World Health Organization approved the use of sodium cyclamate in 1977 asa sweetener for foods and beverages in more than 40 countries (Ahmed & Thomas, 1992).
In humans and animals, most part of cyclamate normally is eliminated in the feces, but a variable quantity is converted to cyclohexylamine by microorganisms that inhabit colon and caecum (Wills et al., 1981). Cyclohexylamine is rapidly absorbed and excreted by the kidneys. Urinary excretion of cyclamate and cyclohexylamine indicates that little remains in the tissues or body fluids after prolonged administration of high doses (Renwick & Williams, 1972). Furthermore, both can be transported through the placental barrier and thus exposed to the fetus (Pitkin et al., 1969). The cyclamate crosses the placenta and, in the fetal circulation, reaches 1/4 of the maternal concentration, and tends to be found in the liver and kidneys (Pitkin et al., 1970).
Various studies have been completed on laboratory animals seeking to verify the toxicity of sodium cyclamate. Kroes et al. (1977) studying the long-term toxicity of cyclamate, saccharin and cyclohexylamine in 2,400 rats, verified that only cyclohexylamine was found to be embryotoxic when administered at a concentration of 0.5%. Takayama et al. (2000) investigating long-term toxicity and carcinogenicity in monkeys fed cyclamate at 100 mg/kg and 500 mg/kg, five times per week for 24 years, found irregular spermatogenesis and sporadic cases of various malignancies. Sasaki et al. (2002), evaluating the toxicity of sodium cyclamate in kidneys of rats, found that 3 and 24 hours after a single 2.000 mg/kg oral dose of sodium cyclamate, significant damage to the DNA was induced in cells of the stomach, colon, kidney and urinary bladder.
Currently in Brazil, there are various table sweeteners with a base of cyclamate and saccharin, with the best-selling formula in the proportion two parts cyclamate for one part saccharin (Cardello et al., 2001). Assuncao et al. (1994), evaluating the quantity of sweetener consumed daily by 36 diabetic individuals, found that 92% add non-caloric artificial sweeteners to a base of cyclamate and saccharin, in quantities less than the daily acceptable ingestion (DAI) that corresponds to 50 mg/kg of body weight (Oser et al., 1975).
Therefore, a research into the effects of sodium cyclamate on kidneys of rat fetuses becomes relevant, since information does not exist in the literature on morphometry of the proximal or distal convoluted tubule nor on the collecting duct in the period of greatest teratogenicity. Furthermore, the possibility of nephrotoxicity may reinforce the hypothesis that this salt, often utilized as a sweetener, can harm the health of animals and humans.
The objective of this study was to evaluate the effects of sodium cyclamate on the kidneys of rat fetuses, in terms of morphometric alterations in the glomerulus, proximal and distal convoluted tubules and collecting duct.
MATERIAL AND METHOD
10 adult rats were utilized (Rattus norvegicus, Wistar), weighing an average of 238g. The animals were maintained in individual cages and fed with ration pellets and water ad libitum. This study was approved by the Commission on Ethics in Animal Experimentation of Faculdade de Medicina de Sao Jose do Rio Preto, Sao Jose do Rio Preto, SE Brazil.
The females were placed with males for breeding in a proportion of 1:3 during the night, and, by morning, were examined by means of vaginal smear, from which visual evidence of spermatozoids was considered the first day of pregnancy. From the 10th to 14th days of pregnancy, five rats received a daily intraperitoneal injection of sodium cyclamate at a dosage of 60 mg/kg of body weight (Labsynth Ltd., Diadema, SE Brazil) for five days.
On the 20th day of pregnancy, the rats were killed by inhalation of sulfuric ether. By means of an ample incision into the abdomen and uterus, the fetuses were collected and immediately immersed in Alfac fixation solution, left to fix for 5 days. After this period, one fetus was chosen at random from each rat, after which the placenta and umbilical cord were separated. The fetus and placenta were weighed separately on a Labof precision balance (Laboratorium Felszerelese, Budapest, Hungary); the length of the umbilical cord was measured with a millimeter ruler. The fetuses were dehydrated and encased in blocks of paraffin for completion of seriated sections with thickness of 6 [micron]m. Next, they were stained with hematoxylin eosin to enable the morphometric exam.
Glomerular volume was calculated with Hund H500 Wetzlar optical microscope (Helmut Hund Gmbh, Germany), objective of 40x, containing a Leitz Wetzlar (Germany) lucida camera, with final augmentation of 496x. The glomerulus were projected onto white sulfite paper and contoured with a black number two pencil. 20 structures were obtained per animal for a total of 100 structures for each group. To obtain the diameters, major and minor axes (mm) were measured from each one of these images. Mean geometric diameter (M) and glomerular volume (V) were calculated from M= (D.d e V= ((/6). M3
For the morphometric study, karyometry was used to evaluate the nuclear parameters of form and volume, as well as their relations by means of the major and minor diameters of epithelial-cell nuclei of the renal proximal and distal convoluted tubules and collecting ducts. The cuts were analyzed by a Hund H500 Wetzlar optical microscope (Helmut Hund Gmbh, Germany) with an immersion objective (100x), with a Leitz Wetzlar clear camera (Germany). The nuclei were projected into white sulfite paper with a final enlargement of 1,240x. 50 images were obtained from each structure studied for a total of 250 images per group, which were subsequently contoured with a black number two pencil, taking care to consider only the elliptical images. The diameters were obtained by measuring the major and minor axes (mm) of these images. The following parameters were evaluated according to Sala et al. (1994): mean geometric diameter, ratio between major and minor diameters, volume, area, perimeter, ratio between volume and area, eccentricity, coefficient of form, and index of contour.
Statistical comparison between control and treated groups was performed by the non-parametric Mann-Whitney test (Conover, 1999).
Results for the quantitative parameters fetal weight, placental weight, umbilical-cord length for control rat fetuses and those treated with sodium cyclamate are shown in Table I.
Glomerulus. Results regarding glomerular volume of the kidneys for control and treated groups are displayed in Table II. It is notable that the glomerular volume of the treated group was significantly greater in comparison to that of the control.
Proximal convoluted tubule. Results corresponding to nuclear diameters of the proximal convoluted tubules in the kidneys for the control group and that treated with sodium cyclamate are presented in Table III. In the treated group, it was observed that the minor and major nuclear diameters of cells in the proximal convoluted tubules were significantly larger than those of the controls. Furthermore, significant increases were found in volume, area and perimeter of nuclei as well as the ratio of volume to area in the epithelium of the proximal convoluted tubule in the treated animals. However, there was no significant change in the nuclear form between groups.
Distal convoluted tubule. In comparison to the controls, the nuclei of the epithelium of the distal convoluted-tubules in the group treated with sodium cyclamate presented significant increases in diameter and size, but no alteration in the nuclear form (Table IV).
Collecting duct. Morphometric study of the collecting ducts of rat fetuses treated with sodium cyclamate revealed that the nuclei presented increased diameters (Table V), which implies augmented perimeter, area and volume and volume-area ratio in these nuclei. Yet the nuclear form in the collecting ducts was shown to be unaltered compared to the control group.
In this study, sodium cyclamate produced in the kidneys of rat fetuses statistically significant morphometric changes in the glomerulus, distal and proximal convoluted tubules, and collecting duct, suggesting nephrotoxicity. In rats treated with sodium cyclamate, the increase in nuclear size in cells of the epithelial tissue was significantly greater than the control group.
Table I. Mean values of fetal weight (g) and placenta weight (g), and umbilical cord length (cm) of control rats fetuses and treated with There is a statistically significant increase in glomerular volume (30.21%) in the kidneys of rat fetuses treated with sodium cyclamate when compared to the control group (p = 0.007). This finding was similar to that verified by Portela (2003), who investigated fetal toxicity in the use of the pharmaceutical product amicacine and found an increase of 40.69% in the glomerular volume of treated rats. According to the author, this result could be due to the proliferation of glomerular cells by an increase in mitotic activity.
Pereira & Mandarim-de-Lacerda (2001), researching quantitative alterations in the rat glomerulus due to inhibition of nitric-oxide synthesis by means of administering L-arginine methyl ester (L-Name), reported a 33% increase in glomerular volume.
The karyometric analysis showed that values for mean nuclear diameter in cells of proximal and distal convoluted tubules as well as the collecting duct in rats treated with sodium cyclamate were significantly greater than those of their controls. These results differ from those obtained by Ravagnani (2000) who reported that 66 rat fetuses treated with acyclovir, known as a nephrotoxic agent, presented a significant reduction in the nuclear diameter of these structures.
Analysis of volume, area and perimeter of cell nuclei in distal and proximal convoluted tubules and also the collecting duct of the kidney in rat fetuses treated with sodium cyclamate showed that there is significant difference in the dimensions for the treated group compared to the controls. These results were the opposite of those obtained by Ravagnani, who obtained significant reductions in these parameters in the acyclovir-treated group compared to the controls.
The variables eccentricity, coefficient of form and index of convolution did not present a significant difference between treated and control groups, in contrast with the data of Ravagnani for cell nuclei of the distal and proximal convoluted tubules as well as the collecting duct, which showed greater values for the fetuses exposed to acyclovir.
Mean fetal body weight was statistically less for the group treated with sodium cyclamate (mean = 2.29g) compared to that of the controls (2.93g). These results confirm data of Liedtke et al. (1995) who, analyzing the effects of cyclophosphamide in rat fetuses, also reported a lower mean body weight in animals treated with this substance (mean = 1.92g) in relation to that of control group (4.07g). This parameter appears to be an indicator of fetal change or embryotoxicity due to both substances.
Various drugs and chemical agents cross the placenta rapidly and reach the fetus. However, the mechanisms involved in these processes are little understood. According to Pitkin et al. (1970), sodium cyclamate crosses the placenta and, though the fetal circulation, attains 1/4 of the maternal concentration, being found primarily in the liver and kidneys.
Mean placental weight of rats treated with sodium cyclamate (mean= 0.31 g) was significantly less than that of control group (mean= 0.47g). This result corroborates that obtained by Matheus et al. (1994) who verified that placentas of rats treated with cyclophosphamide showed lesser mean weights. Smaller placentas probably present reduced blood flow, resulting in considerable fetal hypoxia, which may cause retardation of intrauterine growth (Emmanouilides et al., 1972). Although the purpose of this work had not been to test the hypothesis that sodium cyclamate crosses the placenta, the results suggest that this phenomenon could have occurred.
As to the length of the umbilical cord, although the difference was not significant, the umbilical cords of fetuses from datos treated with sodium cyclamate were shorter than those of the control group. This measure is related to fetal growth in general. The cord grows in response to tensor forces that depend on both fetal movement and intrauterine space, a situation which occurs during early development. Thus, the umbilical cord, as noted in this study, can indicate reduction of fetal movement during gestational period (Miller et al., 1981). In fact, Moessinger et al. (1982), evaluating the length of the umbilical cord in rat fetuses placed outside the uterus, showed that the length of these also was greater than that of the control fetuses coming to term.
This work opens perspectives for new researches regarding morphological changes produced by sodium cyclamate such as duration time of the cellular modifications produced, the reversibility or irreversibility of such modifications and their repercussions on cellular physiology of on the implicated tissues. Consequently, new studies are necessary that seek to confirm whether the effects of the use of sodium cyclamate are temporary or permanent. Considering the lesion caused by sodium cyclamate in treated rat fetuses, it can be concluded that this substance, much used by the world population, should be restricted during pregnancy especially in the third trimester, which in the human species corresponds to the period of formation of nervous tissue, in other words, that of the greatest teratogenicity.
Based on the results obtained, it can be concluded that there is a significant increase in glomerular volume and nuclear size of cells of the distal and proximal convoluted tubules and also the collecting ducts in the kidneys of rat fetuses treated with sodium cyclamate. These morphometric alterations suggest nephrotoxicity.
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Table I. Mean values of fetal weight (g) and placenta weight (g), and umbilical cord length (cm) of control rats fetuses and treated with sodium cyclamate. Mann-Whitney test. Parameter Control Treated Ucalc P[U] Fetal weight 2.93 2.29 0 * 0.004 Placenta weight 0.47 0.31 0 * 0.004 Umbilical cord length 2.12 2.00 7ns 0.155 * Significant difference p = 0.01 Table II. Mean values of glomerular volume ([micron][m.sup.3]) of control rats fetuses (C) and treated with sodium cyclamate (T). Mann-Whitney test. C T 42723.87 76897.08 50873.53 51942.88 60369.97 67076.22 50973.16 88445.77 47913.00 77906.75 x = 50570.71 x = 72453.74 C x T p[U]=0.007 * Significant difference p = 0.01 Table III. Mean values of karyometric parameters of cell nuclei (n=50) of proximal convoluted tubules of control rats fetuses and treated with sodium cyclamate. Mann-Whitney test. Parameter Control Treated Ucalc P[U] Longest axis 10.13 11.57 0 * 0.004 Shortest axis 7.58 8.88 1 * 0.008 Geometric mean axis 8.73 10.11 0 * 0.004 Ratio D/d 1.37 1.37 9ns 0.274 Volume ([micron] [m.sup.3]) 380.96 557.53 1 * 0.008 Area ([micron] [m.sup.3]) 61.73 81.14 0 * 0.004 Perimeter ([micron]m) 28.01 32.33 0 * 0.004 Ratio V/A 5.82 6.74 0 * 0.004 Eccentricity 0.64 0.61 5ns 0.075 Shape factor 0.96 0.97 8ns 0.210 Contour index 3.62 3.63 10ns 0.345 * Significant difference p = 0.01 ns = not significant Table IV. Mean values of karyometric parameters of cell nuclei (n=50) of distal convoluted tubules of control rats fetuses and treated with sodium cyclamate. Mann-Whitney test. Parameter Control Treated Ucalc P[U] Longest axis 10.15 12.17 0 * 0.004 Longest axis 6.89 8.28 0 * 0.004 Geometric mean axis 9.40 10.00 6ns 0.111 Ratio D/d 1.51 1.51 12ns 0.500 Volume ([micron] [m.sup.3]) 337.50 544.69 0 * 0.004 Area ([micron] [m.sup.2]) 56.49 79.56 0 * 0.004 Perimeter ([micron]m) 27.08 32.49 0 * 0.004 Ratio V/A 5.55 6.67 0 * 0.004 Eccentricity 0.70 0.71 12ns 0.500 Shape factor 0.94 0.94 12ns 0.500 Contour index 3.68 3.67 10ns 0.500 * Significant difference p = 0.01 ns = not significant Table V. Mean values of karyometric parameters of cell nuclei (n = 50) of collecting duct of control rats fetuses and treated with sodium cyclamate. Mann-Whitney test. Parameter Control Treated Ucalc P[U] Longest axis 10.91 12.73 1 * 0.008 Longest axis 7.56 9.09 0 * 0.004 Geometric mean axis 9.03 10.72 0 * 0.004 Ratio D/d 1.50 1.42 9ns 0.274 Volume ([micron] [m.sup.3]) 425.74 669.40 0 * 0.004 Area ([micron] [m.sup.2]) 66.21 91.39 0 * 0.004 Perimeter ([micron]m) 29.33 34.57 0 * 0.004 Ratio V/A 5.55 6.67 0 * 0.004 Eccentricity 0.68 0.67 10ns 0.345 Shape factor 0.94 0.95 8ns 0.210 Contour index 3.67 3.64 8ns 0.210 * Significant difference p = 0.01 ns = not significant