Isotretinoin


121

Isotretinoin: action on mice tooth
germs and palate

Eleny Balducci Roslindo1
Vanessa Camila da Silva2
Alessandra Rezende Peris3
Karina Gonzales Silvério4

1Assistant Professor - Department of Morphol-
ogy – Faculty of Dentistry at Araraquara –
State University of São Paulo-UNESP
2Graduate student, MSc in Periodontics
Program - Faculty of Dentistry at Araraquara
– State University of São Paulo-UNESP
3Graduate student – Faculty of Dentistry at
Araraquara – State University of São Paulo-
UNESP
4Graduate student, MSc, PhD in Periodontics
Program – Faculty of Dentistry at Araraquara
– State University of São Paulo- UNESP

Received for publication: April 16, 2002
Accepted: November 27, 2002

Correspondence to:
Profª. Drª. Eleny Balducci-Roslindo
Rua Humaitá, 1680,   Caixa Postal 331 -
Centro
CEP 14801-903    Araraquara – SP Brasil
e-mail: eleny@foar.unesp.br
Fone/Fax: 55 11 5575-1337

Abstract
Vitamin A and its derivates, acid retinoic, tretinoin and isotretinoin, are
currently used in dermatological treatments. The administration of
high doses of this vitamin provokes malformation in the following
systems: central nervous, cardiovascular, skeletal, brain and face. This
study compares the tooth germs of the first maxillary and mandibular
molars and the palate of fetal mice submitted to isotretinoin during
organogenesis. Twelve 60-day-old female Mus musculus were divided
into two groups on the 7th day of pregnancy: treated group – 2 mg
isotretinoin per kg body wight, dissolved in vegetable oil, was admin-
istered orally from the 7th to the 13th day of pregnancy; control group
– vegetable oil in an equivalent volume was administered orally for the
same period. On the 16th day of pregnancy, the females were sacrificed,
the fetuses were removed and their heads were amputted. After stan-
dard laboratory procedures, 6-mm-thick serial sections were stained
with hematoxylin and eosin for light microscopy examination. The
results showed that the control group had closed palates with no traces
of epithelial cells and two fetuses of treated group had insufficient
development in the lateral palate processes, having lack of fusion in
the midline. The first molar germs of the isotretinoin-treated animals
showed delayed development compared to the control animals. In
conclusion, isotretinoin was shown to be  toxic causing retardation of
tooth germs and palate development.

Key Words:
isotretinoin, tooth germ, development, palate.

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Braz J Oral Sci. 1(3): 121-125                                                                                          Isotretinoin: action on mice tooth germs and palate

Introduction
Vitamin A acts on vertebrate organisms during ontogenesis
and pre- and post-birth cellular differentiation process,
reproduction, normal growth, maintenance of visual function,
regulation and proliferation of tissue epithelial structures1.
Besides this, derivates of vitamin A as retinoids were
observed at the endogenous form in embryonic mouse
mandible being important in the formation of the dental lamina
at the beginning of odontogenesis and in defining the
morphology of incisors and molars6.
Among the synthetic derivates of vitamin A, we may find
isotretinoin (13-cis-retinoic acid), currently indicated in the
treatment of ichthyosis, illness of Darrier, psoriasis, acne,
carcinoma of basal cells and in the prevention of
carcinogenesis. In spite of its clinical effectiveness, it may
cause some undesirable side effects such as: depression,
psychotic behaviors, hydrocephaly and facial malformation.
Teratologic effects of vitamin A include malformation of the
central nervous, cardiovascular, skeletal, and brain systems
and face deformation2. The use of isotretinoin by women
during the first quarter of pregnancy caused defects in the
members as well as gastric and hepatic systems of children3.
It was also noticed that the critical period was between the
2nd and 5th weeks after conception.
In the rats, mice and monkeys, the placental transference of
13-cis-retinoic acid during the pregnancy period was
observed when the clorio-allantoic placenta was well
developed7. When another derivate of vitamin A, isotretinoin,
was systemically administered at the concentration of 1 mg
per kg body weight to pregnant mice, the studies8,9 verified
that this drug produced structural alterations in the fetus as
alterations at the development of the tooth germ and in the
palate closure. Besides these alterations, studies4,5  observed
total and partial fusion of the maxilary incisors and the
mandibular molars, agenesis of the incisors, formation of
diastema in the incisors region, supernumerary teeth and
absence of temporomandibular articulation in mice fetuses.
However, as metabolism in rodents is more active than in
humans,  it is necessary to correlate the exposition period of
the embryo to the administered drug doses and its
concentration in order to obtain the results that would
confirm the described alterations in the literature.
Considering the lack of information on isotretinoin effect in
human organism, the aim of the present work was to study
the palate closure and tooth germ development of the
maxillary and mandibular first molars in mice fetuses submitted
to isotretinoin during the organogenesis in the concentration
of 2 mg per kg.

Material and Methods
Twelve 60-day-old female Mus musculus (albino Swiss
variation) primiparous mice were used. The animals were fed
with granular ration and water ad libitum. The gestation

period was determined by identifying the vaginal plug as
day “zero” of pregnancy after the mice mated during the
night, in the proportion of two females for each male of the
same species.
On the 7th day of pregnancy, the females were divided into
two groups: treated group – 2 mg of isotretinoin per kg body
weight, dissolved in vegetable oil, was administered orally,
once a day, from the 7th to the 13th day of pregnancy; control
group – vegetable oil in an equivalent volume was
administered orally for the same period.
After 16 days of pregnancy, the females of both groups were
sacrificed by ip injection of 10% chloral hydrate (4 ml/100g
body weight). Following an abdominal incision, the uterus
was removed and placed on a Petri dish containing saline
solution. Fetuses were removed, weigh and measured in the
brain tail direction and examined macroscopically for the
identification of possible morphologic alteration. After
macroscopic analyses, fetuses had their heads removed and
fixed in Bouin solution, decalcified by Morse10 solution for
histological analyses of palate and first mandibular and
maxillary molar germ development. The heads were embedded
in paraffin, and serial 6-mm-thick slices were stained with
hematoxylin and eosin. The slides were analyzed by light
microscopy.

Results
MACROSCOPIC FINDINGS
All specimens in number of 6 fetuses for each female mouse
were histologically analyzed using serial 6um frontal incisions
of similar depth and thickness (Figures 1 and 6).
The macroscopic evaluation of the sacrificed fetuses on the
16th day of fetal life did not show external malformation, re-
absorption or dead born mice. However, it was verified that
the isotretinoin-treated fetuses presented average body size
and weigth (1,13 g) inferior to the control fetuses (1,42 g).

MICROSCOPIC FINDINGS
Control Group
The palate was totally fused without epithelial cells remaining
in the fusion line of the palate process with the nasal septum
(Figure 1).  Bone trabeculae with variable size and thickness
growing toward the midline, containing in its surface
osteoblasts was also observed (Figure 2). Tooth germs in
cap stage were connected to oral epithelium through the
dental lamina. Figures 3 and 4, demonstrate that the enamel
organ was constituted by outer epithelium formed with a
layer of cubic cells and the stellate reticulum formed by
polygonal cells. The intermediary stratum was constitued
by elongated cells. Adjacently, the internal ephitelium in the
cusp area was formed by highly cylindrical cells arranged
perpendicularly to the dental papilla forming the pre-
ameloblasts, and in the other areas the cells were short and
cylindrical. In the peripheral cells of the dental papilla,



123

especially in cusp regions, short cylindrical cells were
identified, the future odontoblasts arranged in parallel
exhibiting central rounded nuclei. In the central region, it
was possible to observe a loose connective tissue, highly
cellularized, containing ectomesenchymal cells, fibroblasts,
and blood vessels of small diameter. The dental follicles
involving the whole embryo were constituted by loose
connective tissue rich in cells arranged in parallel rows with
dental organ surface. In the lateral areas of the dental organs,
neoformed thin immature bone trabeculae was noticed, with
large medullar space, showing osteoblasts in their periphery
and osteocytes identified in the interior of mature trabeculae.

Treated Group
The microscopic analyses revealed that the majority of
the specimens had fused palates however, two fetuses
p r e s e n t e d  i n s u f f i c i e n t  f u s i o n  i n  t h e  l a t e r a l  p a l a t e
processes, having lack of fusion in the midline (Figure 5).
The fetuses with fused palates showed an absence of
b o n e  t r a b e c u l a e  w i t h  c e l l u l a r  d i f f e r e n t i a t i o n  a n d
m u l t i p l i c a t i o n  m a i n l y  a r o u n d  t h e  b l o o d  v e s s e l s
characteristics of the beginning and end of the ossification
process (Figure 7).
The tooth germs of the first molars were found in different
development stages including the cap or initial bell-
shaped phase for some inferior embryos (Figures 8, 9,
and 10). In the cap phase (Figure 9), three different layers
of cells were observed. The external layer was constituted
by cuboid cells forming the external epithelium. The inner
l a y e r,  o r  i n n e r  e p i t h e l i u m ,  w a s  c o n s t i t u t e d  b y  s h o r t
cylindrical cells in the cusp regions, and in the others
r e g i o n s  b y  c u b o i d  c e l l s .  O n  t h e  c e n t r a l  l a y e r,  a n
agglomeration of rounded and indifferentiated cells that
would form the stellate reticulum and the intermediary
stratum with discrete deposition of intracellular substance
between stellate reticulum cells beginning a differentiation
of enamel organ cell layers was noticed.
Figures 8, 9 and 10, demonstrate a higher concentration
of ectomesenchymal cells and small diameter vessels in
the concavity of the enamel organ. Beyond the dental
f o l l i c l e  a r o u n d  t h e  d e n t a l  g e r m  w a s  c o n s t i t u t e d  b y
connective loose tissue highly cellularized with fibroblasts
arranged in parallel rows, except in the region of dental lamina.
Adjacent to the dental organ, in the majority of specimens,
thin irregular immature bone trabeculae with wide medullar
spaces, showing osteoblasts with spherical nuclei located
on its surface was noticed. Osteocytes in small number
were seen in the interior of mature bone trabeculae.

Figure 1 – Control Group – Frontal slice of the head of embryos.
H&E. Magnification: 32X.

Figure 2 – Control Group – Close palatal processes in the midline
and formation of bone trabeculae. H&E. Magnification: 125X.

Figure 3 – Control Group – First maxillary molar germs. Cap
Phase. H&E.   Magnification: 125X.

Braz J Oral Sci. 1(3): 121-125                                                                                          Isotretinoin: action on mice tooth germs and palate

Figure 4 – Control Group – First mandibular molar germs. Cap
Phase. H&E. Magnification: 125X.



124

Discussion
Odontogenesis in mice begins at the 9th day of fetal life and
the formation of dental bud occurs at the beginning of the
12th day11,12. In this period, drugs that have teratogenic
potential, such as vitamin A and derivates, may cause the
formation of the supernumerary bud and molar substitution
by incisors in the molar region, as verified by Kronmiller et
al.5 when they administered all-trans-retinoic acid in the
exogenous form in the period previous to the formation of
the dental lamina.
Studies13,14 that administered isotretinoin and its metabolie
4-oxi-isotretinoin to pregnant female mice, showed that these
drugs may exert inhibitory effect on the migration of the
neural-crested cells, besides promoting reduction of
members and palate opening. Ritchie & Webster15 carried
out some studies with the purpose of determining the
teratogenicity of isotretinoin and showed in vitro that use
of this drug in the concentration of 500 ng/ml for a period of
a least 6 hours of exposure before the migration of the cells
in the neural crest, was enough to induce severe defects on
the second visceral arch in most of the exposed embryos.
These results are likely to explain the two fetuses with palate

Braz J Oral Sci. 1(3): 121-125                                                                                          Isotretinoin: action on mice tooth germs and palate

Figure 7 – Treated Group – Closed palatal processes in the midline
and no formation of bone trabeculae. H&E. Magnification: 125X.

Figure 6 – Treated Group - Frontal slice of the head of embryos.
H&E. Magnification: 32X

Figure 8 – Treated Group – First maxillary molar germs. Bell-
shaped phase. H&E.   Magnification: 125X.

Figure 9 – Treated Group – First mandibular molar germ. Bell-
shaped phase. H&E.  Magnification: 125X.

Figure10 – Treated Group – First mandibular molar germ. Cap
phase. H&E.  Magnification: 125X.

Figure 5 – Treated Group – Palatal processes having lack of fusion
in the midline. H&E.  Magnification: 125X.



125

opening, caused by the insufficient development of lateral
palatine processes, when 2 mg of isotretinoin per kg of body
weight was administered to pregnant female mice, during
the period between the 7th and the 13th day of gestation.
Concerning odontogenesis, we know that tooth development
is related to a series of complex inductive interations between
two embryonic tissues, the epithelium in the first branchial
arch and the ectomesenchyme derived from neural crest cells.
In the gastrula phase of embryonic development, the
ectoderm and the mesoderm respond more to the excess of
vitamin A, and the endoderm responds less 16,17. During
organogenesis, vitamin A produces an interruption in the
cephalic ectomesenchyme or alters significantly cell
properties18. Subsequently, the action of vitamin A has more
negative effects on bones, cartilages, and dental organs than
positive effects on growth in general.
It is knwon that vitamin A is mobilized in liver stocks and it is
taken to the peripheral tissues by means of highly regulated
transportation. Takahashi & Smith19 verified that in rat
embryos, during the pregnancy period, the presence of
protein connects to retinol (RBP) and that the transportation
system works in the intra-uterine life of mammals. The
presence of those proteins in fetal organs was also studied
by Lorente & Miller20, who verified that a direct action of
vitamin A on tissues and organs is more probable than
placental alterations.
In the literature we did not find histological works on
placentas showing the systemic action of isotretinoin.
However, we observed in this study some placental
alterations in the treated group (not shown). These alterations
indicated an unfavourable development for their gestational
age. Since the development of teeth can be influenced by
nutritional status21,22 the placental alterations would no be
responsible for the embryological alterations caused by
isotretinoin.
The data obtained from the fetuses from the treated group
suggest that isotretinoin, even when used in low dose, 2 mg
per kg of body weight, but administered from the 7th to the
13th day of pregnancy may cause insufficient development
and growth of the lateral palatine processes, delay in the
development of the first mandibular and maxillary tooth
germs, and lower size and weight, when compared to the
fetuses from the controlled group.
When Balducci-Roslindo9 et al. administered 1 mg per kg of
isotretinoin in female mice during the period from the 7th to
the 13th day of pregnancy, they noted the same alterations in
fetal development and growth and in their bone formation.
We can conclude that isotretinoin had an adversed effect on
the size and weigth of the fetuses examed. Additionaly, this
drug may affect the formation of bone tissue in the palatine
process and a delay in the development of the first mandibular
and maxillary molars.

Acknowledgements
The authors would like to thank the financial support
provided by PIBIC/CNPq (1998-1999).

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Braz J Oral Sci. 1(3): 121-125                                                                                          Isotretinoin: action on mice tooth germs and palate