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Luciana Barros Sant’Anna and Darcy de Oliveira Tosello Department of Morphology, Faculty of Odontology, State University of Campinas (UNICAMP), Piracicaba, SP, Brazil.
The consumption of alcohol during pregnancy causes fetal congenital malformations, including craniofacial and orodental defects, as a result of interference with normal embryonic development. In this work, we examined the effects of alcohol on tooth development and enamel formation in rats. Alcohol was administered to female rats in the drinking water starting at a concentration of 1% followed by weekly increases to 5%, 10%, 15%, 20% and 25%. In the seventh week, the rats were mated and continued to receive 25% alcohol until delivery. On postnatal day 5, three offsprings of each mother were killed and their hemimandibules removed, processed and embedded in araldite. Sections 1 μm thick were cut and stained with 1% toluidine blue and histomorphometric analysis of the dental germ and enamel matrix was done. During the postnatal period, the body weights of the offspring from treated dams were significantly smaller than the controls. In addition, the relative volumes of the tooth germ and enamel matrix were always smaller in the offspring of dams treated with alcohol. These results indicated that the ingestion of alcohol during pregnancy interfered with the development of the tooth germ and the secretion of the enamel matrix.
Key words: Enamel formation, ethanol, molar, rat, tooth, tooth germ
calcification of the dentin matrix [8]. These studies The ingestion of high levels of alcohol during show that the influence of alcohol during gestation pregnancy causes serious birth defects because of the varies according to the drinking pattern, the period disruption of normal embryonic development, with and duration of ingestion, and the doses and routes fetal alcoholic syndrome being the most devastating of these defects [3]. Fetal alcoholic syndrome is Amelogenesis can be affected by various chemical characterized mainly by the retardation of pre- and agents, including tetracycline [21] and nicotine [15]. postnatal growth, CNS deficiencies and a particular Alcohol also produces ultrastructural changes in secretory ameloblasts of the tooth germ of mini-pig The effects of alcohol on craniofacial and fetuses after intrauterine exposure to this substance, orodental development include the formation of and results in an abnormal secretory function [14].
small teeth, hypoplastic maxilla [4] and enamel, In this study, we used histomorphology and and other dental anomalies [11]. Studies in mice histomorphometry to assess the development of the have shown a delay in the eruption of the incisors enamel matrix and tooth germ of the first mandibular (S.A. Tomazela-Herndl, personal communication) molar in the offspring of female rats treated with and reduced dimensions of the cranium and jaw teratogenic doses of alcohol during pregnancy. [5,8,9]. Alcohol also causes cellular alterations in the basal epithelial cells of the tooth germ in the MATERIAL AND METHODS
bud stage and in the inner enamel epithelium during odontogenesis [1]. The ingestion of 20% alcohol Two-month-old female Wistar rats weighing 150- before and during gestation delays cell proliferation 230 g were housed individually in standard, clear plastic in the tooth germ during the bud stage, as well as breeding cages. The rats were fed a commercial diet (Purina rat chow) and water ad libitum until the beginning of the experiment. After a week of acclimation, the rats Correspondence to: Dr. Luciana Barros Sant’Anna were randomly assigned to either the treated group (given Departamento de Morfologia, Faculdade de Odontologia de Piracicaba ethanol, n=25) or the control group (n = 17). The treated (FOP), Universidade Estadual de Campinas (UNICAMP), Av. Limeira 901, CP 52, CEP 13414-903 Piracicaba, SP, Brazil. Tel: (55) (19) 3412-5200, group was given ethanol (Merck & Co., Inc., Whitehouse Fax: (55) (19) 3421-0144. E-mail: Station, NJ, USA) added to the drinking water at a Braz. J. morphol. Sci. (2005) 22(3), 155-159 starting concentration of 1% (v/v). This concentration was obtained in a similar orientation in all histological was subsequently increased at weekly intervals to 5%, sections of the control and treated groups. The tooth germ
10%, 15%, 20% and 25% in a manner that allowed the and enamel matrix were evaluated microscopically and rats to become accustomed to and accept the taste of alcohol, thereby avoiding abstinence or a loss of interest in the solution. Control rats received alcohol-free water, and both groups were fed the same solid diet. The treated The histomorphometric analysis consisted of determining rats were given alcohol for six weeks before mating the three-dimensional measurements of anatomical structures (pre-fertilization period). After this period, female rats from bidimensional images of histological sections by were mated overnight with non-alcoholic males. The geometric and statistical analyses [12]. The parameter used presence of sperm in the vaginal smear, detected on the for histomorphometric analysis was the volume density following morning, was defined as day 0 of pregnancy. (V ), which represents the fractional volume occupied by Six pregnant females from each group (controls and an object in a determined structure.
treated) were used. During gestation, a 25% alcohol Two-dimensional profile images of the tooth germ solution was given to the treated group while the control were captured with a light microscope equipped with a group received alcohol-free water (post-fertilization video camera connected to a computer. Volume density period). All of the rats were weighed weekly and the food measurements of the tooth germ and enamel matrix were and alcohol consumption was measured daily. This study obtained using an image analyzer, KS 400 (Kontron was approved by the institutional Committee for Ethics in Electronik, Germany) that elaborated a specific test Animal Experimentation (CEEA/IB/UNICAMP).
system consisting of a square lattice of 70 test-points (test area) superimposed over the tooth germ image to allow stereological counting. This system captured all of the tooth The body weight of each offspring was recorded germ within the hemimandible. The final magnification during the postnatal period and, on the fifth postnatal day, used was 40x in which each square measured 4x104 μm2 three offspring were superficially anesthetized with ether (total of 200 μm)2. When transformed into mm3, each and killed by cervical dislocation for subsequent removal point was equivalent to 8x10–3 mm3.
of their hemimandibles. The tissues were fixed in 2.5% Prior to the histomorphometric analysis, the number glutaraldehyde and 2% paraformaldehyde for 6 h and then of observations required to provide an adequate sample demineralized in 5% EDTA. The hemimandibles were size for reliable measurement and subsequent statistical sectioned transversally in the mesial and distal faces of the analysis was determined using Chalkey’s method [2]. A first mandibular molar (Fig. 1). With this procedure, only pilot study was done on some sections to determine the the tooth germ of the first mandibular molar remained. enamel matrix volume density that should be used under Following this, the tissues were dehydrated in a graded our study conditions. With a test system composed of 70 series of acetone, infiltrated and embedded in araldite. points (tested area), six histological sections per animal, Parasagittal sections 1 μm thick were obtained from the i.e., 420 points corresponding to six tested areas per tooth first mandibular molar in a mesio-distal direction using a Porter Blum MT-2C ultramicrotome and stained with Once the sample size (six serial sections at 10 μm 1% toluidine blue. Using this procedure, the tooth germ intervals) was established, the V of the tooth germ and enamel matrix was determined in 18 treated and control hemimandibles. The volume density of the tooth germ (V ) and enamel matrix (V ), expressed in mm3/mm3, where P is the number of points coincident upon the tooth germ of each rat, P is the number of points coincident upon the enamel matrix from each animal, and P is 6 x Figure 1. Schematic drawing of the hemimandible
with the tooth germ of the first mandibular molar on the fifth post-natal day showing the localization of two transversal sections (a and b) in the mesial and distal
The results were expressed as the mean + SD. The normality of the data was tested using Shapiro-Wilk’s W Braz. J. morphol. Sci. (2005) 22(3), 155-159 test [16] followed by Student’s t test to compare the results DISCUSSION
for the control and treated groups. Values of p≤0.05 Various methods for studying the effects of alcohol consumption have been reported in the literature and differ in the species, ethanol concentration, experimental groups, duration of During pregnancy, the average volume of alcohol ethanol administration and periods of exposure used ingested by the treated group was 24.78 g/kg/day [1,8,9]. In the present study, oral administration was (34.04 ml of 25% alcohol per rat). The initial weight used because it is more similar to human exposure. (281 ± 15.94 vs 246 ± 9.70) and the ended weight of During pregnancy, the average volume of alcohol pregnant females rats (368 ± 22.53 vs 288 ± 15.71) ingested by the treated group was 24.78 g/kg/day were significantly reduced in treated group, despite (34.04 ml of 25% alcohol per rat). In most studies maintaining a healthy appearance; the average on gestational alcoholism, this concentration is weight gain of the controls was approximately 50% sufficient to produce teratogenic effects in the greater than that of the treated rats (Table 1). The gestational period of the treated mothers was longer As show here, exposure to ethanol before and than that of the control rats, and the pups of treated during gestation affected the gestational period, litter dams weighed 21% less than the control pups at size (number of offspring/dam) and body weight of birth (4.77 ± 0.23 g vs 6.04 ± 0.82 g) and on the fifth the offspring of alcoholic dams, a finding that agreed postnatal day (7.56 ± 0.47 g vs 9.58 ± 1.57 g).
with other studies [8-10]. Alterations in maternal Histomorphologic analysis in the control group nutrition represent an important source of interactive revealed normal structural characteristics of the factors in fetal alcoholic syndrome in most animal tooth germ in the appositional stage (Fig. 2). models. However, studies in rodents have shown that Ameloblasts appeared as tall, columnar epithelial ethanol directly affects the development of embryonic cells, with elongated basal nuclei, and vacuoles tissue and results in a low birth weight, even when the were occasionally seen in the supranuclear region of nutritional status of the dams is maintained to allow these secretory cells. The apical cytoplasm contained normal weight gain during pregnancy [9,20]. Our dense, round granules of different sizes. The stratum findings are consistent with other studies [5,7] which intermedium showed a single layer of cells with have shown that alcoholic dams lose body weight large, round nuclei (Fig. 3A) and, in the alcohol- during pregnancy and have a smaller number of treated group, there was a reduction in the thickness offspring compared to isocalorically fed dams. These of the enamel matrix (Fig. 3B). This was confirmed results suggest that alcohol directly affects capacity by histomorphometric analysis, which showed that to bear offspring, regardless of the nutritional status. the volume density of the enamel matrix and the tooth germ was significantly lower in the offspring of treated dams compared to control dams (~50% and ~12% lower, respectively) (Fig. 4). Table 1. Gestational period and weight gain in female
rats, number of offspring per dam, and weight of litters at
birth and on post-natal day 5.
Figure 2. Photomicrograph of the first mandibular mo-
lar on the fifth post-natal day showing the structural char- acteristics of the tooth germ in the appositional stage. E
enamel, D – dentin, P – pulp. Toluidine blue staining. Bar
The values are the mean ± SD. *p≤0.05 compared to the controls.
Braz. J. morphol. Sci. (2005) 22(3), 155-159 In the present study, pair-fed control rats were not embryonic tissues and organs, and leads to an used because there is no evidence of morphological overall reduction in animal growth [7,13,17]. In differences in the mandibular size [7,9] and tooth agreement with this, alterations in tooth size and development [8,10] of offspring from ad libitum and faulty enamel formation have been reported in human fetal alcoholic syndrome [1,4,8,10]. In the The chronic ingestion of ethanol during gestation present study, histomorphometric analysis showed delays the development and differentiation of that the tooth germ volume at the appositional stage was significantly smaller in litters from treated dams compared to the controls, indicating that alcohol interfered with the process of differentiation and the formation of the tissues in this phase. This finding agrees with Hernandez-Guerrero et al. [10], who also observed that the tooth germs of offspring from ethanol-treated mice were morphometrically smaller and that the maternal ingestion of alcohol reduced the expression of epidermal growth factor (EGF) in mouse molar dental follicles, which contributed to the reduction in tooth germ size. Campos and Duranza [1] reported cellular alterations in the tooth germ of mouse molars in the bud and cap stages; the most important alterations were found in the inner enamel epithelium of the tooth germ. Since these cells will differentiate into ameloblasts and begin producing enamel, these modifications could explain the reduction in the enamel matrix seen in our experiments. Guerrero [8] reported retardation in the tooth bud formation in ethanol-treated mice. In these animals, the mandibular bud of the first molar consisted of thick cuboidal epithelial cells, whereas in the controls the tooth germ in bud stage consisted of cylindrical cells at the periphery and polygonal Figure 3. Photomicrograph from the lingual vertent region
of the tooth germ. A – control group, B – treated group.
The mechanism by which alcohol affects the tooth AM – ameloblasts, E – enamel, P – ameloblastic process-
es, SI – stratum intermedium, arrowhead – granules in the
germ is not yet fully understood. Alcohol may affect apical cytoplasm, arrows – terminal bars and (*) dentino-
a variety of processes, including interaction with enamel junction. Toluidine blue staining. Bar = 16 μm.
the membranes of developing cells and alterations in the growth factors and cellular metabolism needed for normal growth. Alcohol is a potent teratogen that affects the proliferation, migration and differentiation of neural crest cells, thereby retarding the development of cellular structures that are dependent on morphogenesis [17]. The cells of the neural crest differentiate in several directions during embryonic development and give rise to various structures, including most of the embryonic connective tissue of the facial area that contributes to the development of the teeth [18]. Since ethanol inhibits EGF receptors [6], it is Figure 4. Mean values ± SD of the volume density (mm3/
possible that EGF may be prevented from exerting mm3) of the enamel matrix and tooth germ of offspring on its function during ameloblastic differentiation, the fifth post-natal day in the control and treated groups. Braz. J. morphol. Sci. (2005) 22(3), 155-159 hence delaying cellular activity and reducing 4. Church MW, Eldis F, Blakley BW, Bawle EV (1997) deposition of the enamel matrix. This suggestion Hearing, language, speech, vestibular, and dentofacial agrees with Guerrero [8] who reported that the disorders in fetal alcohol syndrome. Alcohol. Clin. Exp.
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delayed calcification of dentin at postnatal day 5. Edwards HG, Dow-Edwards DL (1991) Craniofacial 1.5 in alcohol-treated animals resulted in reduced alterations in adult rats prenatally exposed to ethanol. cellular activity and delayed functions because Teratology 44, 373-378.
of the effects of alcohol in the early stages of 6. Gerhart MJ, Reed BY, Veech RL (1988) Ethanol inhibits embryonic development. An ultrastructural study some of the early effects of epidermal growth factor in
vivo. Alcohol. Clin. Exp. Res. 12, 116-118.
of tooth germs in mini-pig fetuses from alcohol- 7. Giglio MJ, Vieiro M, Friedman S, Bozzini CE (1987) treated dams showed that the mitochondria of Effect of prenatal ethanol exposure on the growth of rat secretory ameloblasts had an abnormal shape, mandible skeletal units. J. Biol. Buccale 15, 211-216.
with the deposition of paracrystalline material in 8. Guerrero JCH (1990) Morphologic effects of maternal the matrix and the abnormal deposition of stippled alcohol intake on skull, mandible and tooth of the intercellular material. These changes suggested offspring in mice. Jpn. J. Oral Biol. 2, 460-469.
9. Hernández-Guerrero JC, Ledesma-Montes C, Loyola- that the administration of ethanol during pregnancy Rodríguez JP (1998) Effects of maternal ethanol intake on influenced the secretory function of the ameloblasts second alcoholic generation murine skull and mandibular size. Arch. Med. Res. 29, 297-302.
The results of the present study, and those of 10. Hernández-Guerrero JC, Portilla-Robertson J, Ledesma- other investigations [13,16], have shown that the Montes C, Ponce-Bravo S, Miranda-Gómez A, Arias-Rivera EM (1996) Immunoexpression of epidermal tissue responses to the ingestion of alcohol during growth factor in odontogenesis of the offspring of pregnancy are not uniform. Hence, we observed alcoholic mice. Bol. Estud. Med. Biol. 44, 25-30.
that the reduction in volume density was more 11. Jackson IT, Hussain K (1990) Craniofacial and oral pronounced in the enamel matrix than in the manifestations of fetal alcohol syndrome. Plast. Recon- tooth germ as a whole, which may indicate that str. Surg. 85, 505-512.
12. Mandarim-de-Lacerda CA (1995) Métodos Quantita- ameloblasts were more susceptible to ethanol than tivos em Morfologia. EDUERJ: Rio de Janeiro.
other cells during the period analyzed. In support of 13. Phillips DE, Krueger SK (1992) Effects of combined this conclusion, Phillips and Krueger [13] stated that pre- and postnatal ethanol exposure (three trimester cells with intense metabolic activity show a specific equivalency) on glial cell development in rat optic nerve. Int. J. Dev. Neurosci. 10, 197-206.
In conclusion, alcohol intake during pregnancy 14. Römert P, Matthiensen ME (1988) Changes of secretory ameloblasts in mini-pig fetuses exposed to ethanol in reduced the development of the tooth germ and vivo. J. Dent. Res. 67, 1402-1404.
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rats currently in progress should contribute to our 16. Shapiro SS, Wilk MB, Chen HJ (1968) A comparative study of various tests for normality. J. Am. Stat. Assoc. 63,
understanding of the cellular effects of maternal 17. Sulik KK, Johnston MC, Webb MA (1981) Fetal alcohol syndrome: embryogenesis in a mouse model. Science ACKNOWLEDGMENTS
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