Ultrastructural Pathology

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4NQO-Induced Rat Tongue Carcinoma: An

Ultrastructural Study

Marilena Vered DMD, Sylvie Polak-Charcon PhD, Tania Babushkin PhD & Dan
Dayan DMD, MSc

To cite this article: Marilena Vered DMD, Sylvie Polak-Charcon PhD, Tania Babushkin PhD &
Dan Dayan DMD, MSc (2008) 4NQO-Induced Rat Tongue Carcinoma: An Ultrastructural Study,
Ultrastructural Pathology, 32:5, 199-205, DOI: 10.1080/01913120802034645

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Published online: 10 Jul 2009.

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 Ultrastructural Pathology, 32:199205, 2008
Copyright r Informa Healthcare USA, Inc.
ISSN: 0191-3123 print /1521-0758 online
DOI: 10.1080/01913120802034645

4NQO-Induced Rat Tongue Carcinoma: An

Ultrastructural Study
Marilena Vered, DMD
Department of Oral
Pathology and Oral
Medicine, School of
Dental Medicine, Tel
Aviv University, Tel
Aviv, Israel
Sylvie Polak-Charcon, PhD Tania Babushkin, PhD
Institute of Pathology, Institute of Pathology,
The Chaim Sheba The Chaim Sheba
Medical Center, Medical Center, Tel
Tel Hashomer, Israel Hashomer, Israel and
Department of Life
Science, Unit of Electron
Microscopy, Bar Ilan
University, Israel
Dan Dayan, DMD, MSc
Department of Oral
Pathology and Oral
Medicine, School of
Dental Medicine, Tel
Aviv University, Tel
Aviv, Israel

ABSTRACT
The 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinoma, in which the carcinogen is
administered systemically in drinking water, is the most comparable animal model to the develop-
ment of human oral carcinoma. This is the first study to report the ultrastructural changes in this
model. The most significant changes were observed in the carcinoma cells at the invasion front and
included unique modifications in the basal lamina, presence of micropinocytotic vesicles (plasma-
lemmal caveolae), and emergence of cytoplasmic microfilaments featuring a parallel arrange-
ment. The microfilaments, in both appearance and organization, were consistent with contractile
microfilaments. These observations may be the morphological reflection of the phenotypic modifi-
cations occurring within the carcinoma cells, approaching smooth muscle differentiation.

Keywords: 4NQO, tongue carcinoma, microfilaments, smooth muscle differentiation

Oral cancer consistently ranks as one of the top ten
cancers worldwide [1]. Lately, an alarming increase
in the incidence of oral cancer, particularly tongue
cancer, in patients under the age of 40, has been
reported [2]. Despite the recently reported drop in the
overall death rate from cancer, the estimated survival
rate (B50%) and number of deaths from oral cancer
remain virtually unchanged [3].
Chemically induced oral cancer in animal models is
aimed to assist us to further understand the events lead-
ing to the development of oral cancer in human patients.
The changes at the light-microscope level and histo-
chemistry, immunohistochemistry, and modifications in
the genetic profile during experimental oral cancer have
been vastly investigated and thoroughly documented
[46]. However, the ultrastructural changes have been far
less studied. In regard to tongue cancer, only two studies
in the English literature were published in which the
carcinogenic agent was administered by topical applica-
tion [7, 8]. Nevertheless, Otsubo and Kameyama used
7,12-dimethylbenzo(a)anthracene (DMBA) and hamsters
[7], while Jiang et al., employed 4-nitroquinoline 1-oxide
(4NQO) and rats [8]. The main findings in these studies
included presence of cytoplasmic projections of the basal
and intercellular aspects of the cancer cells, considerable
decrease in the number of desmosomes, occurrence of
atypical cytoplasmic tonofilaments, increased intercellular
spaces, and changes in the basal lamina ranging from
absence to discontinuity to normal appearance and to
increased thickness or multilayering.

Received 4 October 2007; accepted 31 December 2007

Address correspondence to Prof. Dan Dayan, Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University,
Tel Aviv 69978, Israel. E-mail: ddayan@post.tau.ac.il
199
 Ultrastructure of 4NQO Carcinoma in Rats

The 4NQO tongue carcinogenesis model, in which the grades of ethanol, and washed in 0.1 M buffered
carcinogen is systemically administered in drinking cacodylate. The specimens were fixed in 2.5% glutar-
water, is recognized as the most reliable simulator of oral aldehyde in 0.1 M buffered cacodylate, postfixed in
cancer development in human patients [5, 9, 10]. In this 1% osmium tetroxide for 1 h, dehydrated in a series
model, small amounts of carcinogen are ingested over a of increasing ethanol concentrations, and finally
long period of time, during which premalignant lesions embedded in epoxy resinAgar mix (Agar Scientific,
of increasing severity develop and culminate with the Essex, UK) for 2 days at 601C. From the blocks,
appearance of a true malignant tumor of squamous cell semithin sections were prepared and relevant areas
carcinoma (SCC). The pattern of the clinical, histopatho- were selected for ultrathin sections and stained with
logical, and nuclear changes of the rat tongue carcinoma uranyl acetate and lead citrate. Examination was
have been found to be equivalent to those described in performed in a Jeol 1200 EX transmission electron
human patients with a fair degree of consistency. In our microscope (Jeol, Peabody, MA, USA).
previous study on this model, we observed that evolu-
tion of tongue SCC was accompanied by a sudden sig-
nificant increase in the stromal myofibroblasts. The cells
were identified by routine immunohistochemical proce-
RESULTS
dure with an antibody against alpha-smooth muscle Figure 1 shows representative regions of normal
actin (a-SMA), a cytoskeleton protein characteristic of tongue (A) and SCC (B) as seen on the hematoxylin
myofibroblasts [13]. In addition, at the tumorstroma and eosin-stained slides, corresponding to tissue
interface we have observed a-SMA-stained cells that blocks submitted for electron microscope examination.
displayed an epithelioid morphology and were remark-
ably similar to SCC cells. Determining whether these cells
were myofibroblasts with an epithelioid morphology or
SCC cells positive for a-SMA was problematical. The
present study was aimed at examining the ultrastructural
changes in the systemic 4NQO tongue SCC model to
look for support for the presence of a-SMA immuno-
reactivity in the tumor cells. In addition, we compared
findings in relation to the corresponding ultrastructural
studies in human oral carcinoma.

MATERIALS AND METHODS

Study Group
Formalin-fixed, paraffin-embedded blocks of the control
(N 7) and experimentally 4NQO-Induced SCC (N 9)
were used from a previous study [10]. In the experi-
mental group, 4NQO (Fluka AG, Switzerland) was
dissolved in drinking water at a final concentration of
0.001% and supplied for a total period of 28 weeks.
Water was replaced twice weekly with freshly prepared
solution. Rats were euthanized by the administration of
CO2, the tongues were dissected and a longitudinal
midlingual incision was made. All experimental proto-
cols involving animals were approved by the Animal
Committee of the Sackler Faculty of Medicine, Tel Aviv
University, and conformed to procedures described in
the Guiding Principles for the use of Laboratory Animals.

EM Preparation
Areas of squamous cell carcinoma (SCC) and of
normal tongue epithelium were marked on the tissue
blocks, respectively. These were extracted, deparaffi-
nized in xylene, rehydrated in a series of decreasing
Figure 1. Representative fields of normal tongue epithelium
(A) and SCC at the tumor connective tissue interface (B)
(hematoxylin and eosin,
400 original magnification).
200
 M. Vered et al.

Figure 3. Normal tongue: Basal cells are usually of an even size

Figure 2. SCC islands surrounded by myofibroblasts (a-SMA and form a regular row of cells. Epithelial-connective tissue
positive, brown color). Within the central SCC island, a- interface is remarkable for a highly folded appearance and an
SMA-stained epithelioid cells are seen (a-SMA antibody, intact basal lamina. CT, connective tissue; BL, basal lamina.
AEC method, original magnification
400).

4NQO-induced Tongue SCC

Figure 2 represents a section from a case of SCC that
was immunohistochemically stained with antibody
against alpha smooth muscle actin (a-SMA) (clone
1A4, 1:100, Dako A/S, Denmark), as described in
detail in our previous study [10]. The brown stain is
indicative of immunoreactivity of the actin cytoplas-
mic microfilaments to the antibody. Positive stain is
highlighted in several stromal spindle-shaped cells
that occasionally surround SCC islands. However, in
the center of the photomicrograph, a cluster of
positively stained cells is seen amid a tumor island.
These cells display an epithelioid morphology and
cannot be distinguished from the adjacent SCC cells.
Identification of this type of cell urged us to examine
the ultrastructural characteristics of SCC cells to
investigate the possible presence of actin microfila-
ments in these cells.
Ultrastructurally, the cells at the periphery of the tumor
islands were of varying sizes and possessed remark-
ably pleomorphic nuclei (Figure 6). The epithelial
connective tissue interface was irregular. At the basal
aspect of most of the peripheral neoplastic cells, a
band of basal lamina-like substance could be observed.
Drawing an imaginary line that follows the basally
located external lamina from sequential adjacent
peripheral cells would highlight a ragged barrier
between the tumor and the connective tissue compart-
ment. At a higher magnification it became obvious
that this basal band of external substance was part of a
well-developed, continuous external lamina that sur-
rounded each SCC cell (Figure 7). At the lateral cell
borders, the external lamina of adjacent cells merged

Control Tongue Epithelium

Electron microscopic examination revealed cuboidal to
low-columnar basal cells that were of a similar size
(Figure 3). They possessed large, centrally located nuclei
with occasional nucleoli. The epithelialconnective
tissue interface was highly folded, denoting an inter-
digitating appearance. The basal lamina was of a regular
width, continuous and tightly following the outline of
the finger-like cytoplasmic projections in the basal
aspect of the basal cells (Figure 4). Hemidesmosomes
and well-developed desmosomes were present. The
spinous cells displayed an abundance of desmosomes
around their plasma membrane attaching them to the
neighboring cells (Figure 5A). Desmosomes occasionally Figure 4. Normal tongue: hemidesmosomes (H) and desmo-
were associated with tonofilaments (Figure 5B). somes (D).
201

Figure 5. Normal tongue: Abundant desmosomes (D) (A)
associated with tonofilaments (T), (B) are a constant feature
of the spinous layer.
Figure 6. SCC, front of invasion: irregular-sized, congested
peripheral cells form a ragged epithelial-connective tissue
interface. At the basal aspect of these cells, a band of basal
lamina-like material is present. CT, connective tissue.
together to form an intercellular thicker band, ulti-
mately creating a network in which the neoplastic cells
Ultrastructure of 4NQO Carcinoma in Rats
Figure 7. SCC, front of invasion: cells are individually sur-
rounded by an external lamina (arrows).
were embedded. (Figure 8A). On further magnification
of the intercellular region, some electron-dense plaque-
like structures were seen on the plasma membrane
aspect of the cells on both sides of the external lamina,
but they lacked the architecture of typical desmosomes
(Figure 8B). Alternating with these attachment plaques,
a number of uncoated micropinocytotic vesicles (plas-
malemmal caveolae) were observed (Figure 8B). The
cytoplasm of the SCC cells contained numerous micro-
filaments running parallel to each other, sometimes
aggregated into oriented bundles, along which several
electron-dense areas (focal densities) were identified
(Figure 8B). These organized microfilaments aggregated
into long bundles/band-like structures and usually
aligned parallel to the plasma membrane and the long
axis of the cells. They were seen in many SCC cells, but
they were more conspicuous in those at the invasion
front (Figure 9). The distribution of the microfilaments
was not uniform, their presence being more conspicuous
in some areas of the cytoplasm than in others.
Other intracellular organelles, such as mitochondria,
Golgi apparatus, and rough endoplasmic reticulum
were occasionally observed in the neoplastic cells but
their frequency did not differ remarkably from the
cells in the control samples.
DISCUSSION
This is the first study in the English literature of the
ultrastructural changes in the 4NQO rat tongue
carcinogenesis model, where the carcinogen was
administered systemically, in drinking water. The
importance of this study lies in the observation that
this particular model reproduces in the closest way the
stages of evolution of oral cancer in human patients.
Cellular features corresponding to the state of maturation
of the epithelial layers were observed in the normal
202
 M. Vered et al.
Figure 8. SCC, intercellular space: remarkable thick band
of external lamina (asterisk) (A). At a higher magnification
(B), membranal electron-dense plaque-like structures, micro-
pinocytotic vesicles (arrow) and numerous cytoplasmic,
parallel microfilaments (M) with occasional electron-dense
areas (focal densities) (dotted arrow) are seen.
epithelium. Well-developed systems of hemidesmosomes
and desmosomes were always seen. Cytoplasmic pro-
jections in the basal region of the basal cells were bor-
dered by an intact and continuous basal lamina. This is in
accordance to the well-illustrated findings in the control
tongues in the studies of Jiang et al. [8] and Otsubo and
Kameyama [7]. These projections might have a role in
increasing the surface of the epithelialconnective tissue
interface and providing a stronger bond between these
203
Figure 9. SCC, invasion front: cytoplasmic bundles of
microfilaments ran parallel to each other and to the long axis
of the cells and are directed towards the invasion pathway.
CT, connective tissue.
compartments. The major ultrastructural differences
found in this study between squamous cell carcinoma
(SCC) cells and their normal counterparts consisted of
unique changes in the organization of the basal lamina,
presence of micropinocytotic vesicles, modifications in
the cell-to-cell and cell-to-matrix junctions, and emer-
gence of cytoplasmic microfilaments featuring a parallel
arrangement.
The tumorconnective tissue interface had an irregular
outline. There were numerous, tightly packed, vari-
able-sized peripheral, disarrayed SCC cells. A band of
basal lamina-like substance at the basal aspect of each
of these cells contributed its part in forming a ragged
barrier between the tumor and the connective tissue
compartment. This basally located band was part of a
continuous, prominent external lamina that sur-
rounded each of the tumor cells. At the remaining cell
borders, the external lamina of adjacent cells united to
form a network in which the neoplastic cells were
embedded. As no regular adhesion systems of hemi-
desmosomes and desmosomes were identified, it

seems that this external lamina served as a means of
connecting between the tumor cells. The many
micropinocytotic vesicles found in the cytoplasm in
the vicinity of the cell membrane may be evidence
for the existence of an active transport of products
synthesized within the cells and required for the
establishment of the external lamina.
Studies on experimental tongue cancer performed
with topically applied carcinogen, either in rats [8] or
hamsters [7], reported changes in the basal lamina at
the tumorconnective tissue interface that ranged from
normal ultrastructural morphology, to discontinuity,
and to prominent focal thickening/multilayering. Cell-
to-cell adhesion in the topically applied carcinogen
model was usually provided by desmosomes located
at the lateral cell borders, although they were fewer
than in the normal tongue epithelium [8]. It seems that
our finding of an external lamina network encircling
the tumor cells mainly at the invasion front is exclusive
for the development of tongue carcinoma following
systemic administration of 4NQO.
Evidence of focal basal lamina multilayering/reduplica-
tion/thickening has been previously reported in experi-
mental carcinogenesis [8, 11] as well as in human oral
SCC [12, 13]. In general, this can be explained in terms of
either an increased turnover with synthesis of additional
cellular/extracellular products by the cells exposed to
micro-environmental insults, or may reflect the cellular
response to the dysplastic changes [14]. An additional
theory for the excessive production of external lamina
relates to the possibility of locomotion of the SCC cells
in a centrifugal pattern [12]. Areas with thickening of
external lamina may present the trailing edge of the
cancer cells. As these cells move forward, the trailing
edge will leave behind the original external lamina and
form successive layers of new lamina. On the other hand,
thinning and absence of this lamina probably represent
the leading portion of cancer cells, which are in the
process of active locomotion. Further support to this is
provided by the observation that following local inva-
sion cells may cease migration and produce a distinct
basal lamina, and that its disappearance seems restricted
to foci of ongoing tumor invasion [15]. Accordingly, in
the present study, where the external lamina network
was of various thickness it could be assumed that SCC
cells were in different phases of migration.
Active locomotion of migrating SCC cells is probably a
central contributing factor to their invasiveness. This
can be facilitated mainly by the presence of a con-
tractile microfilament system [16]. Contractile cyto-
plasmic microfilaments have been identified in
experimental carcinogenesis of the dermis [17]. Our
results are the first to show occurrence of this type of
microfilaments in the rat systemic 4NQO-induced
SCC. The microfilaments displayed either a parallel
Ultrastructure of 4NQO Carcinoma in Rats
pattern or an aggregation into bundles that ran parallel
to the cell membrane. This organization conformed to
the morphological appearance of the contractile com-
ponents observed in cells with a recognized contractile
potential, such as myofibroblasts [18]. The microfila-
ments in SCC cells might be consistent with actin
filaments, thus explaining our previous observation of
epithelioid, actin-stained cells at the tumorconnective
tissue interface [10]. This, together with the presence of
the micropinocytotic vesicles, sited in a pattern remi-
niscent of smooth muscle cells [19], further supports
the phenotypic changes occurring in the SCC cells,
which approach a smooth muscle differentiation. In
the study by Jiang et al., in which 4NQO was topically
applied on the rat tongues, atypical tonofilament
bundles that appeared as linear structures in the
perinuclear and peripheral cytoplasm of the neoplas-
tic cells were reported [8]. In that study, they were
assumed to indicate either a disturbance of epithe-
lial maturation or an association with cytoplasmic
streaming in locomotive tumor cells. Recognition of
a considerably increased number of contractile micro-
filaments (e.g., actin) in human SCC cells (including
those of oral epithelium) as compared to their normal
counterparts was done by Shenk [16] and Gabbiani
et al. [20, 21] about 3 decades ago. Furthermore, Harris
investigated spindle cell carcinomas of the skin and
breast and found that the morphological changes were
accompanied by ultrastructural evidence of partial or
complete mesenchymal metaplasia of the carcinoma
cells [22]. Chen and Harwick also reported presence
of cytoplasmic microfilaments in oral SCC, but unfor-
tunately their findings were not properly illustrated [12].
Our identification of contractile microfilaments may be
the morphological reflection of the invasive potential
of the neoplastic cells. It can be assumed that at any
given time SCC cells could open a new invasive front
as evidenced by the uneven appearance of the tumor
connective tissue interface. This would depend, for
example, on the number of contractile microfilaments
present in the cytoplasm of the SCC cells. As the
number of microfilaments ranged among the SCC cells
from none to many, it seems that those with a higher
concentration of contractile microfilaments will be
burdened with the task of drawing their neighboring
cells that lack cytoplasmic microfilaments. The net-
work of external lamina surrounding the SCC cells
might serve as an intercellular adherence system to
transmit the locomotion force from cell to cell in the
absence of desmosomes. Changes in the thickness of
the external lamina in certain regions could indicate
the movement of the most potently invasive SCC cells
at a given time point.
The present study was the first to describe the ultra-
structural changes in the SCC cells in the systemic
4NQO rat tongue model. Extensive production of
204
 M. Vered et al.
basal lamina-like material and presence of contractile
cytoplasmic microfilaments parallel the changes
described in oral SCC in human patients. This further
emphasizes the potential of this animal model to
contribute to our understanding the mechanisms of
carcinogenesis in humans. Classification of the cyto-
plasmic microfilaments and their functions in this
model should be performed to further explain the
evolution of the malignant phenotype.
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