The effect of adriamycin on rat incisor one day after administration.

THE ANATOMICAL RECORD 211:17-23 (1985)
The Effect of Adriamycin on Rat Incisor One Day
After Administration
A.C. KARIM
Department ofAnatomy, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3
ABSTRACT
The effect of adriamycin on rat incisor was investigated 1 day after
administration. Rats were injected intravenously with adriamycin a t a dose of 5 mgl
kg body weight and sacrificed by perfusion with a 2.5% phosphate buffered glutaraldehyde solution. The principal effect of the drug on the incisor was the production
of cell degeneration. This was extensive in the apical region, being present along
the entire periphery of the dental papilla. In sections examined more incisally, cell
degeneration gradually disappeared from the labial portion of the incisor but was
present in the lingual portion. This degeneration of cells was not present a t the site
where mature odontoblasts had differentiated on the lingual surface of the pulp
chamber. It appears that the affected cells were early preodontoblasts and the
precursors of preodontoblasts. However, as these cells became more differentiated
they apparently became more resistant to the drug’s effect.
Osteodentin resembles bone because of the cellular
inclusions present within the matrix. The latter appears
less dense than that of normal dentin. This may be due
to the large amount of ground substance normally found
in osteodentin (Takuma et al., 1967),and the presence of
fewer collagen fibers (Takuma et al., 1967; Karim and
Eddy, 1984). Unlike dentin, osteodentin is atubular
(Takuma et al., 1967)and is deposited continuously until
the pulp is completely obliterated (Karim and Eddy,
1984). The cells that are responsible for normal dentin
production differentiate into odontoblasts from the mesenchymal cells of the dental papilla under the organizing influence of the inner dental epithelium (Bhussry,
1976).Radioautographic studies have demonstrated that
the odontoblasts participate actively in the formation of
the dentin matrix (Weinstock and Leblond, 1974; Josephesen and Warshawsky, 1982). These studies have
demonstrated protein synthesis in the odontoblasts after
3H-proline administration. This protein synthesis was
first observed in the rough endoplasmic reticulum and
in the Golgi apparatus. It then appeared to migrate from
the Golgi zone to the odontoblastic process in the form
of dense granules, which then emptied into the extracellular collagenous matrix.
It has been proposed that osteodentin formation in the
rat incisor, seen after the administration of cyclophosphamide (Koppang, 1973) and vincristine (Stene, 19781,
was the result of a n injury to preodontoblasts and their
precursors. Although this had never been demonstrated,
it was further suggested that abnormally differentiated
pulp mesenchymal cells would replace these damaged
cells and begin the production of osteodentin (Stene and
Koppang, 1980; Koppang, 1981). Recently, adriamycin
was shown to cause osteodentin formation in rat incisors
(Karim and Eddy, 1984). The present study was undertaken to determine whether injury to preodontoblasts
and precursor cells in the lesion was present in the rat
0 1985 ALAN R. LISS, INC
incisors after administration of adriamycin. This effect
is of significant interest in view of the hypothesis that
osteodentin formation may be the result of such a lesion,
and would provide a basis for future studies on osteodentin formation in this animal model.
MATERIALS AND METHODS
Ten male Sprague-Dawley rats (100 g rt 5) were given
a n intravenous injection of adriamycin at a dose of 5
m g k g body weight. The adriamycin was obtained from
Adria Laboratories of Canada Limited. One day later
the rats were sacrificed by perfusion with a 2.5% phosphate buffered glutaraldehyde solution. Control animals
injected with only physiological saline were treated in
the same manner.
The incisors were removed with the jaws and demineralized in a 4.13% isotonic EDTA solution which was
continuously agitated. After demineralization the incisors were cut into 1=mm =thick cross =sectional segments. Longitudinal segments were also prepared. All
segments were washed overnight in cold phosphate
buffer, postfixed in 1% osmium tetroxide in distilled
water at 4°C for 3 hours, dehydrated in graded concentrations of acetone, infiltrated with acetone-Epon mixtures, and embedded in Epon.
One=pm =thick sections were routinely prepared and
examined with the light microscope. Those segments in
which the lesion was found were further trimmed, and
ultrathin sections were prepared. These sections were
placed on formvar=coated slotted grids and stained with
4% uranyl acetate for 10 min and with lead citrate
(Reynolds, 1963) for 15 min. Observations were made
with a Hitachi HU-12 electron microscope operated a t
75 kv.
Received December 5, 1983; accepted July 20, 1984.
18
A.C. KARIM
Light Microscopic Observations
RESULTS
At the apical end, where the inner dental epithelium
The administration of adriamycin produced a lesion in
which preodontoblasts and pulp mesenchymal cells were reflects a t the cervical loop to form the outer dental
destroyed. This lesion was not localized, but extended epithelium (Fig. 2), cell necrosis was observed along the
from the apical end beyond the point where the initial entire peripheral surface of the dental papilla. This cell
layer of enamel was being deposited incisally. The fol- degeneration manifested itself as dark globules within
lowing results describe this lesion as observed at differ- the sections. In the control sample (Fig. 11, cell degenerent sites along the incisor. Serial and longitudinal ation was not present. Instead, preodontoblasts were
sections through many incisors have failed to show the observed along the labial, lateral, and mesial surfaces of
the dental papilla. Near the apical foramen, mesenchyformation of osteodentin in this study.
ma1 cells were closely apposed to the inner dental epithelium. Further incisally (Fig. 3a), cell degeneration
was minimal within the labial portion of the tooth. At
this site, there was no degeneration in the layer of cells
in close apposition to the inner dental epithelium.
Within the lingual portion (Fig. 3b), this cell layer was
completely removed by necrosis. Necrosis was also seen
within the mesenchymal cells on the periphery of the
dental papilla. However, no necrosis was seen in the
control sample from these sites. Within the labial portion of the incisor (Fig. 4a), odontoblasts were seen lining the periphery of the dental papilla. On the other
hand, within the lingual portion (Fig. 4b), preodontoblasts were seen lining the periphery. Near the cervical
loop, mesenchymal cells were closely apposed to the
inner dental epithelium.
However, at a site where the apical foramen was closed
by the root sheath formation (Fig. 51, cell degeneration
was seen along the periphery of the pulp chamber, and
in the preodontoblast layer within the lingual portion of
the incisors. Within the labial portion no degeneration
was observed (Fig. 6). At this site secretory odontoblasts
were present. Therefore, the cell degeneration was observed at the site of the future cemento-enamel junction
(Fig. 6) and along the entire lingual portion of the pulp
chamber. Examination of control tissue taken a t a n
equivalent site (Fig. 7) has shown no cell degeneration
within either the pulp or the preodontoblast layer.
Further incisally, where secretory odontoblasts were
present on the periphery of the lingual portion of the
pulp chamber, a small region of cell degeneration was
observed. This was located on the periphery of the pulp
chamber and not within the odontoblast layer. When
sections were examined incisal to this point, no degeneration was observed.
Fig. 1. Cross-section through the apical foramen. In this control
sample the inner dental epithelium (IDE) reflects at the cervical loop
as the outer dental epithelium (ODE). The dental papilla (DP) is compact. The preodontoblasts (PO) are located on the periphery of the
papilla and lining the inner surface of the inner dental epithelium.
They do not line the entire surface at this level, but are located mainly
within the labial half of the section. In the lingual half containing the
apical foramen, the cells that are related to the inner surface of the
inner dental epithelium are still immature undifferentiated mesenchyma1 cells of the dental papilla. Toluidine blue. x 160.
Fig. 2. Cross-section through the apical foramen. In this experimental sample, note the extent of the cell degeneration (asterisks)
within the dental papilla (DP). Some necrosis is also seen within the
inner dental epithelium (IDE) and within the stratum intermedium
(arrowhead).The effect of adriamycin at this level removed the entire
population of preodontoblasts. ODE, outer dental epithelium. Toluidine blue. x 160.
EFFECT OF ADRIAMYCIN ON RAT INCISOR
Fig. 3. Cross-section through the apical foramen. One day after
adriamycin treatment. In the labial portion (a), note that the necrosis
(asterisks) is confined to the mesenchymal cells on the periphery of the
dental papilla. At this site the differentiated preodontoblasts (arrowhead) associated with the inner dental epithelium (IDE)are unaffected.
0, odontoblasts; PO, preodontoblasts. In the lingual portion, (b), the
preodontoblast layer is absent, thereby leaving a space adjacent to the
inner dental epithelium. The necrosis (asterisks) is seen within the
rnesenchymal cells on the periphery of the dental papilla. ODE, outer
dental epithelium. Toluidine blue. x 640.
19
Fig. 4. Cross-section through the apical foramen. In these control
samples, the labial half of the section (a)shows preodontoblasts (arrowliead) at the bottom and odontoblasts (0)a t the top, both lining the
inner surface of the inner dental epithelium (IDE). The dental papilla
(DP) has closely packed mesenchymal cells. In the lingual half of the
section (b), note that the preodontoblasts (arrowheads) are lining the
inner surface of the inner dental epithelium (IDE). However, at the
level of the apical foramen, mesenchymal cells are associated with the
inner dental epithelium. ODE, outer dental epithelium. Toluidine blue.
x 200.
20
A.C. KARIM
Fig. 5. Cross-section through the lingual portion of the incisor. One
day after adriamycin treatment. At this site the apical foramen is
closed by the formation of the root sheath (RS). Note that the necrosis
(asterisks) is present within the preodontoblast layer on the inner
surface of the root sheath, and within the peripheral region of the pulp
chamber (PI.Toluidine blue. x 320.
Fig. 6. Cross-section through the incisor. One day after adriamycin
treatment. The necrosis (asterisks) is seen a t the level of the future
cementoenamel junction and along the lingual portion of the incisor,
as seen in Figure 5. At this site, where the initial layer of dentin is
present (arrowheads),there is no necrosis within the labial portion of
the incisor. PO, preodontoblasts; RS, root sheath; 0, odontoblasts; A,
ameloblasts; P, pulp chamber. Toluidine blue. X 320.
Fig. 7. Cross-section through the incisor. In this control sample,
taken at the same level as Figures 5 and 6 , note that there is no
necrosis within the pulp chamber (P).A, ameloblasts; 0, odontoblasts.
Toluidine blue. x 100.
dental epithelium and the preodontoblasts (Fig. 10).
Nearer the labial surface, the preodontoblasts had difThese observations were made from sections that are ferentiated further (Fig. 11)and eventually attained the
fully differentiated state of odontoblasts on the labial
represented in Figures 3a and 3b.
In the lingual portion of the tooth near the cervical surface of the dental papilla (Fig. 12). Collagen fibers
loop, the cells that were degenerating contained large were present between the inner dental epithelium and
electron-dense globules (Fig. 8).These cells had few pro- the odontoblast layer. These fibers also formed large
files of rough endoplasmic reticulum, but there was an bundles between odontoblasts. Examination of control
abundance of free ribosomes within their cytoplasm. At samples taken through the apical foramen has shown
this site a wide space, containing few collagen fibers and immature mesenchymal cells a t the cervical loop. Differa fine flocculent material, separated the degenerating entiated preodontoblasts were near the labial surface of
cells from the inner dental epithelium. Further from the the dental papilla, and fully differentiated odontoblasts
cervical loop, nearer the labial surface (Fig. 9), cell de- were seen on the labial surface. There was no necrosis
generation within the dental papilla was still present. within the pulp or the preodontoblast layer.
Nevertheless, cells were present in close apposition to
DISCUSSION
the inner dental epithelium. Irregularly arranged collaThe presence of necrotic cells in a confined region of
gen fibers were present among these cells.
At the site where there was no cell degeneration, a the apical end of the pulp, as demonstrated in the presfine fibrillar material was present between the inner ent study, appears to be within the area of proliferating
Electron Microscopic Observations
EFFECT OF ADRIAMYCIN ON RAT INCISOR
21
Figs. 8 and 9.Electron micrographs of sections through the lesion
near the apical foramen as shown in Figures 3a and 3b. One day after
adriamycin treatment. In the vicinity of the apical foramen (Fig. 8),
the necrosis (asterisks) is seen in cells containing a n abundance of free
ribosomes. Note also the space between the necrotic cells and the inner
dental epithelium (IDE). Nearer the labial surface (Fig. 9) there are
fewer necrotic cells (asterisks) within the layer of cells associated with
the inner dental epithelium (IDE). Many collagen fibers (arrowheads)
are seen among the cells at this site. DP, dental papilla. X 14,400.
cells, a s seen in the 3H-thymidine studies of Smith and
Warshawsky (1976). These investigators have shown,
through serial sections, that as the cells are carried by
eruption from the apical to the incisal end of the rat
incisor, those on the labial surface mature earlier than
those on the lingual surface. This indicated that imma-
ture cells were present on the lingual surface for a
longer distance from the apical end. In light of the extent of the proliferating zone in the apical end of the rat
incisor (Smith and Warshawsky, 1976) and the toxic
effect of adriamycin on proliferating cells (Barranco,
1975), the present results indicate that mainly imma-
22
A.C. KARIM
Figs. 10 and 11. Electron micrographs of cross-sections through preodontoblasts and inner dental epithelium, as seen in Figure 3a. One
day after adriamycin treatment. There is no necrosis in the preodontoblast layer (PO), which is separated from the inner dental epithelium
(IDE) by a fine fibrillar matrix. rer, rough endoplasmic reticulum. x
14,400.
Fig. 12. Electron micrograph of cross-section through the odontoblasts and inner dental epithelium, as seen in Figure 3a. One day after
ndriomycin trcntmcnt. On the lnbinl surfncc tho odontobhttn (0)nrc
unaffected by the adriamycin. Large bundles of Van Korff's fibers
(arrowheads) are seen passing between the odontoblasts and fanning
out into the initial layer of dentin. IDE, inner dental epithelium; rer,
rough endoplasmic reticulum. x 10,800.
23
EFFECT OF ADRIAMYCIN ON RAT INCISOR
ture dividing cells may be affected. This conclusion is
also supported by our electron microscopic results, which
have shown that morphologically the necrotic cells had
few profiles of rough endoplasmic reticulum and a n
abundance of free ribosomes. They have also shown that
once these immature cells (preodontoblasts and their
precursors) have differentiated to the stage where they
have begun to secrete the first layer of dentin, they are
presumably insensitive to the cytotoxic effect of the drug.
The necrosis seen within the lesion may be due to a n
effect of the drug at the nuclear level (Di Marco et al.,
1971; Wang et al., 1972). On the other hand, since macrophage activity was not observed within the lesion, the
dead cells are probably not removed from the lesion, and
instead, may undergo complete autodigestion through
the activation of hydrolytic enzyme within them (Singal
et al., 1984).
The destruction of preodontoblasts may not be an effect unique to adriamycin. Although it was not previously demonstrated, it has been suggested that the
formation of osteodentin in rat incisor after treatment
with cyclophosphamide (Koppang, 1973) and vincristine
(Stene, 1978) was due to a n injury to preodontoblasts
and possibly their precursors. In the present study, the
lesion extended from the apical end to some point incisally, beyond the closure of the apical foramen. However,
one cannot ascertain from this data whether the bulbous
portion of the odontogenic organ (Smith and Warshawsky, 1976) was affected. Nevertheless, the destruction of the preodontoblasts and precursor cells has
significant functional implications. By way of speculation, if the preodontoblast cell population is not replaced, one would expect a failure in dental hard tissue
formation and in closure of the apical foramen. However, since the incisor is erupting continuously, this
defect eventually would be eliminated, provided that the
stem cell compartment in the bulbous portion of the
odontogenic organ was not affected. On the other hand,
if these destroyed cells are replaced by abnormally differentiated cells, a s was proposed earlier (Koppang,
19731, this would be of signifcant interest in the mechanism of osteodentin formation.
In conclusion, this study has shown that immature
pulp cells a t the apical end of the rat incisor are selectively destroyed after adriamycin administration. The
functional significance of these morphological findings
is presently being investigated.
ACKNOWLEDGMENTS
The author would like to thank Mrs. S.P. Pylypas for
her technical assistance, and Mr. R. Simpson for the
photographic illustrations. This work was supported by
grants from The Manitoba Health Research Council and
The Medical Research Council of Canada.
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