REGULAR PAPER A HISTOMORPHOMETRICAL STUDY OF THE EFFECTS OF ETHANOL ON ENAMEL FORMATION IN RAT MANDIBULAR MOLARS DURING PREGNANCY
Luciana Barros Sant’Anna and Darcy de Oliveira Tosello
Department of Morphology, Faculty of Odontology, State University of Campinas (UNICAMP), Piracicaba, SP, Brazil. ABSTRACT
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 INTRODUCTION
calciﬁcation of the dentin matrix . 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 . Fetal alcoholic syndrome is
Amelogenesis can be affected by various chemical
characterized mainly by the retardation of pre- and
agents, including tetracycline  and nicotine .
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 .
small teeth, hypoplastic maxilla  and enamel,
In this study, we used histomorphology and
and other dental anomalies . 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 . 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: firstname.lastname@example.org
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 . 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 . 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  followed by Student’s t test to compare the results
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. , 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  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  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 . 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 .
Since ethanol inhibits EGF receptors , 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
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