A Study of Salivary Glands of Rats Injected with Actinomycin D
Heun Taik Jhee
Department of Anatomy, Yonsei University, College of Medicine, Seoul, Korea.
Department of Anatomy, Yonsei University, College of Medicine
Seong Soo Han, D.D.S.
Department of Oral Biology, The University of Michigan, School of Dentistry
Since the isolation of actinomycin D from Streptomyces parvallus (Manaker et al.,
1954), its potent cytostatic effects have been widely recognized and numerous
reports have already appeared describing the effects of it and related compounds on
embryonic tissue (Tuchmann-Duplessis and Mercier-Parot, 1958, 1959 and 1960; and
Denis, 1963), various tumors (Gregory et al., 1956; Boric et al., 1957 and 1958;
Oota et al., 1957; Olummer and Walker, 1958; and Awa, 1961) and other normal
tissues (Hackman, 1954; Friederici, 1955; and Bierling, 1960). Meanwhile, the
chemical mechanisms of actinomycin in inducing cytostatic effects have been
clarified to a large extent and experiments have proven that the drug can be used
as an excellent analytic tool for studies of the biosynthesis of proteins,
especially of the role played by the messenger RNA (m-RNA) in this process (Kik,
1960; reich et al., 1961 and 1962; Goldberg and Rabinowitz, 1962; Harbers and
Mueller, 1962, Kahan et al., 1963; Meritz, 1963; Paul and Struthers, 1963; Perry,
1963; and Staehelin et al., 1963). A survey of these articles and others
illuminates the following:
1) m-RNA is produced in the nucleus by the DNA-dependent RNA polymerases;
2) the continued supply of m-RNA is essential for the maintenance of the
functional and structural integrity of polyribosomes;
3) the turnover of m-RNA is relatively fast; and
4) the production of the m-RNA can be inhibited by actinomycin D which combines
specifically with DNA, resulting in the blockage of DNA-dependent RNA polymerase
With respect to the digestive glands synthesizing proteineous enzymes for
secretion, an insult imposed by the administration of actinomycin D would seriously
affect the functioning of the gland which in turn would be reflected in its
structure. In a preliminary communication we have described the weight changes and
cytology of rat parotid and submandibular glands following a single injection of a
sublethal dose of acinomycin D (Jhee and Han, 1964). The present article represents
the final report of the complete study.
MATERIALS AND METHODS
Experimental Design: Twenty eight adult male Sprague-Dawley rats were used. Of
these, twenty on received an intraperitoneal injection of 75 ㎍ actinomycin D
dissolved in 0.5ml of saline. The remaining seven ras served as controls and were
injected with the same volume of saline alone. Animals were fed ad libitum with
regular laboratory rat chows. Following injection, three of the experimental and
one of the control rats were sacrificed on days 1,3,5,7,10,14 and 21. Each rat was
weighted at the beginning of the experiment and at the time of sacrifice, and the
average body weight of the experimental animals was subjected to statistical
Histological Procedures: Upon sacrifice, the right parotid and submandibular
gland were dissected out and weighed. Small pieces of the gland were fixed in
Bouin's solution and in Zenker-formol, double-embedded in parlodion-paraffin and
sectioned at 6 μ.
All sections were routinely stained with hematoxylin and eosin. In addition the
following staining procedures were employed for observing different aspects as
indicated: Unna-Pappenheim's methyl green-pyronin for changes in nucleic acid
contents; PAS-azure Ⅱ for RNA content and cytoplasmic granules; aldehyde
fuchsin-Masson for the general appearance of the parotid gland; and
mucicarmin-hematoxylin and PAS-alcian blue for cytology of the submandibular gland.
The Nucleoli to Nuclei Ratio: In order to obtain a quantitative estimate of the
nucleolar changes of the secretory cells, the ratio of the number of necleoli to
that of nuclei was studied in the following manner. Methyl green-pyronim stained
sections of the parotid gland from block fixed in Zenker-formol were used. Under
oil immersion nuclear counts were made in random areas. From each gland at least
1500 or more nuclei of acinar cells were counted. Of these, the number of nuclei
which contained nucleoli of acinar cells were counted. Of these, the number of
nuclei which contained nucleoli was recorded separately.
The nucleoli to nuclei ratio (No/N) was obtained as follows.
No/N=Number of nuclei containing nucleoli/Number of nuclei x100
RESULTS AND OBSERVATIONS
Body Weights: Table 1 records the changes in average body weight of rats
receiving an intraperitoneal injection of 75㎍ actinomycin D.A gradual and linear
decrease was observed through the first two weeks followed by a partial recovery on
day 21. The control rats kept growing at the rate indicated.
Glandular Weights: The changes in weight of the right parotid and submandibular
glands are shown in Table 2. Of the two glands the parotid suffered a greater and
more rapid loss of weight. By 24 hours the average glandular weight was less than
66% of the control value. As was true with body weight, the lowest point was
obtained on day 14 when the glandular weight decreased to less than 50% of the
control. Again, by the end of the experiment the weight showed a significant
recovery. The high value obtained on day 7 is not consistent with changes in body
and other organ weights.
As expected from the nature of secretory products, the weight of the
submandibular gland was less affected than that of the parotid. The decrease was
more gradual and did not drop as much as the latter. The low point was observed on
day 10 and a moderate recovery was seen by the second week.
Histological and Cytological Changes of the Parotid
The first sign of degeneration was the appearance of focalized areas of nuclear
pyknosis in the parenchyme. Such lesion was recognized as early as 24 hours after
injection and became progressively larger through the first 10 days (Figs. 1 and
2). Despite the progressive damage, some of the acini maintained a near normal
appearance. A reversal of degenerative changes was noted by day 14 and, by the end
of the third week, the gland recovered an essentially normal appearance.
Acinar Cells: Secretory cells of the parotid acini became somewhat smaller
compared to those of the control (Figs. 3,4 and 5). The selective pyknosis of
nuclei of acinar cells was apparent 24 hours following the injection and became
extensive by day 3 (Figs. 4 and 7). The nucleus of such cells was smaller than
control and had an irregular, wrinkled outline. The nucleoplasm has lost the
typical vesicularity and become increasingly condensed (Figs. 7 and 8). By day 7
the nucleus was so dense that only by means of differential staining could the
presence or absence of the nucleoli be determind. Elsewhere the progressive
rarification of the cytoplasm was notable in the affected cells. It seemed to
result from a combination of the decrease in number of secretory granules and the
gradual loss of cytoplasmic basophilia. On day 7 many cells were completely devoid
of basal striations (Fig. 8).
Within the rarified cytoplasm small vacuoles were present, which grew in number
and size (Figs. 5 and8). They were most numerous on day 7 and were often located
adjacent to the nucleus. Sometimes their increase in size appeared to be
facilitated by the coalescence of smaller vacuoles (Fig.8). In some cells the
entire cytoplasm was clear and what seemed to be cytoplasmic aggregates were
scattered throughout the otherwise emptylooking cytoplasm. Nuclei in such cells
were barely recognized as a large clump adhering to the basal plasma membrane.
On day 10 changes indiciating the nuclear recovery were noted in many acinar
cells. The contour of nuclei became rounded and the vesicular appearance returned.
Nucleoli grew larger and prominent. By day 14 nuclei in most acinar cells were
indistinguishable from those of the control (Figs. 3,6,7 and 9). A slight recovery
of the basal basophilia of acinar cells was observed on day 14 and, by day 21, many
acini presented a near normal picture. The only difference between an acinus of a
control rat and that of a 21 day animal of the experimental group was the
relatively small number of secretory granules observed in the latter group (Fig.
The Appearance of Ducts: All ducts of the parotid were essentially not affected.
Throughout the experiment, cells of the intercalated, striated and excretory ducts
appeared normal. However, a difference was noted in the content of these ducts
during the early period. Twenty-four hours after the injection large masses of
stainable materials were present in all portions of the duct system. This could be
stained with eosin after Zenkerformol fixation as well as other cytoplasmic stains.
The eosinophilic materials were cleared by day 3. The cells of the interacinar
connective tissue remained unaltered.
Changes in Number of Nucleoli: To ascertain the results of microscopi observation
of nucleolar changes in quantitative terms, the nucleoli to nuelei ratio was
studied in the manner previously described. This was considered important
especially because of the fact that, during the early period of the experiment,
there was a considerable variation in number of the affected cells from area to
area of the same gland. Talbe 3 summarizes the efforts of quantitation. Although
the initial drop of the ratio during the first 24 hours was similar to weight
changes of the gland, it remained basically unchanged during the first week and, by
day 10, a significant increase was observe. Since the histological picture of
acinar cells was progressively degenerating during this period and the glandular
weight kept dropping, the early quantitaive recovery of nucleoli, independent and
ahead of other cytoplasmic changes, might be important. This is in agreement with
the histological finding in which the nucleolar recovery was seen to proceed that
of the nuclear structure and cytoplasmic basophilia.
Histological Changes of the Submandibular Gland
Acinar Cells: The basic quality of alterations produced in the submandibular
gland was similar to what was observed in the parotid. The degree and extent of
damage, however, was considerably less incomparison to those of the parotid. The
nuclear pyknosis was moderate and the ergastoplasm appeared to persist, although a
significant decrease was apparent (Figs. 11 and 12). A fairly large number of
vacuoles were evident by day 7. As was true in the case of the parotid, an early
nuclear recovery was followed by the return of cytoplasmic normalcy, namely the
disappearance of vaouoles and a rise of basophilia.
As revealed by mucicarmin-hematoxylin staining the mucoprotein content in acinar
cells was notably decreased during the initial phase and did not regain the
staining intensity observable in control rats.
Ducts: Of the extensive duct system only the granulated portion of the striated
duct appeared affected. Compared to changes observed in acinar cells the effect was
rather slow. The ducts remained unchanged during the first 3 days, when the
cytoplasm was undergoing a rapid degeneration (Fig. 13). On day 5 a decrease in
number of the granules was observed in some specimens and by day 7 depletion of the
granules was evident in may cells (Fig. 14). The granules that were still present
assumed irregular size and distibution. The basal striation appeared somewhat less
distinct than in controls. Occasionlly wrinkling of nuclear membrane was observed
in such cells.
A study has been made of the salivary glands of the rat following an injection of
a sublethal dose of actinomycin D. Body weight and weights of the parotid and
submandibular glands showed significant decreases during the first 7 to 14 days.
The loss in weight was the greatest in the parotid gland.
Histological and cytological observations indicated that the changes in secretory
cells occurred in the following order: (1) beginning of nuclear pyknosis with a
rapid decrease in number and size of nucleoli by 24 hours after the injection; (2)
advanced stages of nuclear pyknosis with concomitant decrease in cytoplasmic
basophilia and granules by day 3; (3) apparent vacuolization and rarification of
cytoplasm by day 7; (4) beginning of nucleolar and nuclear recovery by day 10; and
(5) complete cytoplasmic recovery by the end of experiment as evidenced by the
return of basophilia and disappearance of vacuoles.
Paralleling the weight changes, cytological damages were greater in the parotid
than in the submandibular acini. This was thought th be related to differences in
nature of secretory products by the two organs. A descrease in number and
pronounced irregularity in size of the granules in salivary ducts of the
submandibular gland were observed but at a later time than the above mentioned
changes in the acini. The significance of these changes has been discussed in light
of the recently gained knowledge on biosynthesis of nucleic acids and proteins, as
related to the chemical mechanism of actinomycin D in the inhibition of m-RNA