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[CANCER RESEARCH 33, 2498 2506. October 1973]
Acute Changes in the Surface Morphology of Hamster
Tracheobronchial Epithelium following Benzo(a)pyrene and
Ferric Oxide Administration1
Curtis D. Port, Mary C. Henry, David G. Kaufman,2 Curtis C. Harris,2 and Kathleen V. Ketels
IIT Research Institute, Life Sciences Division, Chicago, Illinois 60616
SUMMARY
Acute changes in the surface morphology of hamster
tracheobronchial epithelium were examined by scanning
electron microscopy following intratracheal administration
of ferric oxide or benzo(a)pyrene absorbed to ferric oxide.
Multiple instillation of ferric oxide brought about a loss of
ciliated cells and broad areas of abnormal, enlarged nonciliated cells with roughened or wrinkled surfaces. These
were interpreted as areas of epithelial hyperplasia. Multiple
intratracheal administrations of benzo(a)pyrene and ferric
oxide caused similar changes and, in addition, produced
squamous metaplasia and small foci of markedly abnormal
protuberant cells. These findings are correlated with a pre
viously reported observation obtained by transmission elec
tron microscopy. The results suggest that scanning electron
microscopy is useful in studies of the histogenesis of lung
cancer.
INTRODUCTION
Administration of polynuclear hydrocarbon carcinogens
with paniculate carriers by the intratracheal respiratory
route has been effective in the induction of tumors in the
respiratory tract of Syrian golden hamsters. Saffiotti et al.
(19) have shown that intratracheal instillation of BP3 car
ried on ferric oxide produces squamous metaplasia and
squamous carcinomas in the respiratory tract. The hamster
model now is being used to define the process of squamous
differentiation and to clarify the relationship between squa
mous cell differentiation, metaplasia, and squamous cell
carcinoma. The acute ultrastructural lesions produced as a
result of carcinogen administration (7) and vitamin A defi
ciency (8) have been reported. BP-ferric oxide appears to
cause squamous metaplastic lesions that are different from
those caused by vitamin A deficiency. Defects in cell base
ment membrane, enlarged nuclei deeply indented by cytoplasmic invaginations, and pleomorphic nucleoli appear to
be restricted to the squamous metaplasia induced by BPferric oxide.
Since morphological changes are an integral part of the
neoplastic process, the SEM should be considered an addi
tional tool in its study. The SEM offers distinct advantages
over conventional light microscopes and TEM's. One of
the most useful characteristics of the instrument is its wide
range of magnification which permits the rapid examina
tion of a large surface area with greater depth of focus than
with other light or electron optical systems. For example,
the entire width and length of the trachea can be examined
in 1 specimen.
The specific objective of this study was to evaluate the
applicability of the SEM for the visualization of surface
morphological changes, such as epithelial hyperplasia and
squamous metaplasia, produced by intratracheal instilla
tion of BP-ferric oxide in hamsters. Whenever possible, this
evaluation was correlated to the ultrastructural pathology,
determined by transmission electron microscopy, reported
in the literature.
MATERIALS
AND METHODS
BP with more than 98% purity (Aldrich Chemical Com
pany, Inc., Milwaukee, Wis.) absorbed to ferric oxide (Type
R8089; Pfizer, Inc., New York, N. Y.; particle size dis
tribution: 93% by weight <5 ^m in diameter, 80% <2 /¿m,
and 68% < 1 ^m) or ferric oxide alone, were suspended in
sterile 0.9% NaCl solution. The suspensions were repeat
edly administered by intratracheal instillation to young
adult Syrian golden hamsters4 of both sexes. Although the
BP-ferric oxide mixture was prepared in a 1: 1 ratio of 5 mg
BP and 5 mg hematite, each instillation contained 2.5 mg
BP, as determined by chemical analysis (17), for a total
dose of 25 mg of BP.
The hamsters were divided into 3 groups. Group A con
sisted of 35 hamsters, each receiving 5 mg of ferric oxide.
Each of the 35 hamsters in Group B received 5 mg of ferric
oxide and 2.5 mg of BP. Group C consisted of 20 control
'Supported by Contract 69-2148 from the Lung Cancer Segment, hamsters. In all groups, the intratracheal instillation was
Carcinogenesis, Division of Cancer Cause and Prevention, National Cancer performed 3 times per week (Monday, Wednesday, and
Institute, NIH, Bethesda, Md.
Friday) for a total of 10 instillations. For examination, 3 or
'Lung Cancer Unit, Carcinogenesis. Division of Cancer Cause and
4 hamsters from the 2 treatment groups and 2 hamsters
Prevention, National Cancer Institute, Bethesda, Md. 20014.
3The abbreviations used are: BP, benzo(o)pyrene; SEM, scanning
electron microscope; TEM, transmission electron microscope.
Received November 7, 1972; accepted June 25, 1973.
2498
' Hamsters obtained from S. Poiley, Mammalian Genetics and Animal
Production Section, Drug Research and Development, National Cancer
Institute, NIH, Bethesda, Md. 20014.
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Surface Morphology of Hamster Epithelium
from the control group were anesthetized by an i.p. injec
tion of pentobarbital. The examinations were performed
weekly (Mondays) for a period of 10 weeks.
The abdomen of each hamster was opened on the midline, permitting access to the ventral aorta. The aorta was
severed, the animal exsanguinated, and after the chest was
opened, the lungs and trachea were removed. The trachea
was cannulated to the level of the first cartilaginous ring
and the lungs were expanded, at a pressure of 30 cm of
water, with Karnovsky's paraformaldehyde-glutaraldehyde phosphate-buffered fixative (9). Prior to use, the osmolality of the fixative, about 2010 mOsmoles/kg, was
reduced to approximately 550 mOsmoles/kg by the addi
tion of phosphate buffer. Perfusion continued for at least
2 hr with the lungs completely immersed in fixative. Upon
completion of airway perfusion, the trachea was ligated
and the lungs were floated in fixative.
The trachea and main stem bronchi were removed from
the lungs, washed in distilled water, and dehydrated with
increasing concentrations of alcohol. Amyl acetate then was
substituted for the alcohol, and the tissue was dried by the
critical-point method in carbon dioxide (1). The dried
trachea was sectioned longitudinally, cemented to a copper
stub, and coated with gold in a vacuum evaporator. To en
sure uniform coating the evaporator was equipped with a
rotating turntable. The tissues were examined in a JEOLJSM-2 scanning electron microscope equipped with an
X-ray diffraction apparatus, at 5, 15, and 25 kV.
RESULTS
Normal Tracheobronchial Epithelium. In the untreated
hamster, the tracheobronchial epithelium consisted of cili
ated and nonciliated epithelial cells. Elevations were
formed in the epithelium by the underlying cartilage rings,
with depressions present between rings. The density of
ciliated cells appeared greatest at the edge of elevations
formed by the underlying trachéalrings (Fig. 1) and de
creased toward the middle of the rings and in the spaces
between rings (Fig. 2).
The number of ciliated and nonciliated cells appeared
about equal in the trachea (Fig. 3). The cilia were erect
and free standing, without mucus droplets. The exact de
marcations of ciliated cells usually were obscured by the
cilia and in some preparations by the filiform projections
or villi protruding between the cilia. These projections ap
peared taller than the microvilli of nonciliated cells (Fig. 3).
Nonciliated cells in the trachea were polygonally or pentagonally shaped with raised borders at cell junctions. The
surface of the nonciliated cells was covered with discrete
microvilli which projected slightly into the lumen.
More ciliated than nonciliated cells were seen near the
carina and bronchi (Fig. 4). In the bronchi and lower tra
chea, the nonciliated cells were oblong, had a rounded
surface, and appeared to be squeezed between the ciliated
cells. Cell junctions, characterized by the raised border,
were discernible but not prominent.
Ferric Oxide Treatment. Three intratracheal instillations
of ferric oxide resulted in broad focal areas of nonciliated
epithelium which were distributed randomly along the
length of the trachea (Fig. 5). Individual cells were large
and showed roughened or wrinkled surfaces. These areas,
interpreted as epithelial hyperplasia, were still present after
10 intratracheal instillations. A few ciliated cells were pres
ent in these areas after 3 instillations but were absent after
10 instillations. A transition from areas of epithelial hyper
plasia to more normal appearing epithelium was observed.
Some nonciliated cells in the more normal epithelium ex
hibited raised and roughened surfaces.
Ferric oxide particles, confirmed by X-ray diffraction
(Fig. 6), assumed a variety of shapes, sizes, and surface
structures, and were present at all times during the treat
ment. The tracheobronchial epithelium returned to normal
7 weeks after completion of the treatment.
BP-Ferric Oxide Treatment. Three intratracheal instil
lations of the BP-coated ferric oxide resulted in changes
similar to those observed with ferric oxide alone. In addi
tion, there were regions where nonciliated cells protruded
into the trachéallumen (Fig. 7 and 7/4) or cells which
possessed roughened, wrinkled surfaces. Individual ferric
oxide particles frequently were present in areas of cellular
change.
Six instillations produced a partial to complete loss of
ciliated cells in focal areas of enlarged nonciliated cells. Al
though still randomly distributed, abnormal areas appeared
larger and more numerous than those seen with ferric oxide
alone. Many stages were observed in the degeneration of
ciliated cells; finally, these degenerated cells were shed,
leaving only circular holes in the epithelium. The degenera
tion was demonstrated by shortened cilia and partial col
lapse of the cell surface. In most instances, these cells
showed depressed margins, and many contained shortened
cilia and filiform projections, thereby permitting identifica
tion as degenerating ciliated epithelial cells. (Fig. 8).
Numerous focal areas of enlarged, broad, nonciliated epi
thelial cells showing rough surfaces with raised and
rounded cell margins were present (Fig. 9). These areas ap
peared to be in a transitional stage between basal epithelial
cell hyperplasia and squamous metaplasia (Fig. 10 and
10/1). In the regions of squamous metaplasia, surface cells
appeared to be sloughing from the underlying cells, with
cell margins that curved toward the trachéallumen. The
degree of cell distortion was determined with the help of
stereo-pair photographs (Fig. 11). The underlying cells in
these areas presented surfaces totally different from those
of luminal nonciliated epithelial cells (Fig. 12).
A similar pattern of epithelial hyperplasia and squamous
metaplasia was present after 10 instillations of the BPferric oxide mixture and persisted 7 weeks after the final
intratracheal instillation. These changes, although focal in
nature, involved approximately one-half of the surface of
the trachea. Because of a low density of microvilli on cell
surfaces which resulted in less available surface for the re
flection of electrons, many of the cells in the regions of
hyperplasia appeared black at low magnification.
Ferric oxide particles usually appeared as clumps in
the trachea and bronchi and were identfied readily by Xray diffraction (Fig. 13). In contrast to the individual
particles or small groups of particles observed earlier in
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Port, Henry, Kaufman,
Harris, and Ketels
the experiment, the clumps were numerous and large. Nu
merous holes, marking the location of degenerated ciliated
cells (Fig. 8), were present with white blood cells near or on
top of the holes (Fig. 14).
Large clusters of cells protruding well into the trachéal
lumen were visible I week after the last treatment (Fig.
15). The cells comprising the clusters were large and dis
played the normal surface structure of nonciliated epi
thelial cells, but the cell margins were not well defined.
The groupings were found in areas of epithelial hyperplasia or beginning squamous metaplasia and differed
markedly from other previously observed areas where in
dividual cells were discrete with normal cell margins or,
subsquently, where cell surfaces were rounded.
Abnormal cells of this type, seen 4 weeks after the last
treatment, were in contrast to those observed 1 week after
the last treatment in that, although large, they possessed
a variegated surface. They were not numerous, were ran
domly located, and were observed singly (Fig. 16) or in
clusters (Fig. 17). The usual microvilli were absent, but
were replaced with either short, blunt projections or long,
coiled, interdigitating ridges (Fig. 17). Some cell surfaces
at the base of the enlarged cells also presented a varie
gated surface structure, differing from the more normal
surface structure of adjacent cells (Fig. 17).
DISCUSSION
The results of this study indicate that the SEM is a
useful, appropriate tool for the visualization of preneoplastic surface changes in the respiratory epithelium. Pre
vious studies (7, II, 19, 20) showed that intratracheal in
stillations of BP carried on ferric oxide produces epi
thelial hyperplasia and squamous metaplasia in the res
piratory epithelium of the Syrian golden hamster. In this
study, multiple instillations of ferric oxide produced a loss
of ciliated cells with broad focal areas of epithelial hyper
plasia. Similar changes were observed with the admin
istration of BP-coated ferric oxide. In addition, focal
areas of squamous metaplasia and small foci of protuber
ant cells with abnormal surface structure were observed.
Interpretation of the epitheljal hyperplasia and squa
mous metaplasia rests upon a comparison of SEM photo
graphs and published TEM results (5, 7, 8). In ferric oxidetreated animals, there was widespread focal replacement of
the columnar epithelium with polygonal basal cells. The
transmission electron micrographs showed that the sur
face of the hyperplastic cells was broad, somewhat rounded,
and possessed scant microvilli. Transmission micrographs
of trachea! epithelium following multiple instillations of a
BP-ferric oxide mixture showed areas of squamous meta
plasia and adjacent epithelial hyperplasia (7, 8). The
squamous metaplastic cells were flattened at the surface
of the epithelium, and some edges were separating from
underlying cells and turning toward the trachea! lumen.
Undifferentiated cells, in foci of epithelial hyperplasia,
possessed protruding surfaces with variable numbers of
microvilli.
The experimental protocol used in this experiment and
2500
the TEM experiment (7) was identical, and comparable
results were obtained in terms of time of onset and nature
of the changes. However, the large protuberant single
cells or cell clusters observed by SEM were difficult to in
terpret, as such cells have not been discussed in TEM
studies. The size of the cells and their unusual surface
structure indicate abnormalcy, and they could represent
atypical hyperplastic epithelial cells. While possibly ma
lignant, they could not be judged so, and proper inter
pretation requires further correlation with either light or
transmission electron micrographs. Should they slough
from the epithelium, these types of abnormal cells could
influence exfoliative cytology interpretations (12).
Atypical epithelial hyperplasia and squamous meta
plasia are associated with bronchial carcinoma in humans
(2) and hamsters (19). Exposure of the respiratory epi
thelium to chemical carcinogens causes such changes (2-4,
7, 15, 18, 19). The various stages of trachea! epithelial
alteration, such as basal cell hyperplasia and squamous
metaplasia, can be differentiated by light microscopy and
transmission electron microscopy (8). However, examina
tion of a large area of the respiratory tract by transmis
sion electron microscopy is not feasible. Pease (16) has
estimated that it would require 7.5 years of continuous
microscope use to examine 1 sq cm of ultrathin sectioned
material at x!500. The area shown in Fig. 10 is of com
parable magnification, and illustrates the utility of the
SEM. The SEM permits visualization of the whole
trachea, shows the extent of change, and provides an esti
mate of the surface area involved in the change. At the
same time, at high magnifications, the examination of
small areas of the epithelium and even of individual cells
is possible. Furthermore, stereophotographs and X-ray
diffraction permit visualization of cell distortion and identi
fication of metallic elements. The SEM has been of value
in studies of the respiratory tract (6, 10, 13, 14, 21). In
parallel with light microscopy, the TEM, and exfoliative
cytology, the SEM can be used to obtain a better under
standing of the events leading to cancer of the lung, es
pecially of the relationship between hyperplasia, meta
plasia, and anaplasia.
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1973
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Port, Henry, Kaufman,
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Fig. 1. Normal trachéalepithelium. Ciliated cells form patches or clusters over the ridges created by underlying cartilage rings, x 77.
Fig. 2. Normal trachéalepithelium. Broad areas of nonciliated cells in space between cartilage rings. Two ducts from subepithelial glands are present,
x 77.
Fig. 3. Normal trachéalepithelium. Nonciliated cells are hexagonal or polygonal with raised borders at cell junctions. Ciliated cells are not numer
ous and have filiform projections or villi protruding between the cilia, x 3000.
Fig. 4. Normal bronchial epithelium. Ciliated cells are numerous and partially obscure the rounded, oblong, nonciliated epithelial cells, x 2310.
2502
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..
Fig. 5. Trachéalepithelium following 3 instillations of ferric oxide. Broad focal areas of enlarged nonciliated, epithelial cells with roughened or
wrinkled surfaces, x 770.
Fig. 6. Trachea! epithelium following 10 instillations of ferric oxide. Roughened surfaces of nonciliated cells. Note ferric oxide particle (arrow).
x 2310.
Fig. 7. Trachea! epithelium following 3 instillations of BP plus ferric oxide. Nonciliated cells appear rounded and protrude into trachea! lumen.
x 450. Inset (Fig. 1) is magnified further in Fig. 1A. x 4500.
Fig. 8. Degenerating epithelial cell. Cilia are absent, and cell margins are depressed from surface height of surrounding cells, x 3000.
Fig. 9. Trachéalepithelium after 6 instillations of BP plus ferric oxide. Broad areas of nonciliated epithelial cells with raised and rounded cell mar
gins, x 1000.
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Port, Henry, Kaufman, Harris, and Ketels
IOA
II
Fig. 10. Trachea! epithelium after 9 instillations of BP plus ferric oxide. Focal area of squamous metaplasia with raised cell margins, x 300. Inset
(Fig. 10) is magnified further in Fig. 10/4. Note surface structure of underlying cells, x 1000.
Fig. 11. Trachéalepithelium I week after IO BP plus ferric oxide instillations. Stereo pair of photographs. Degree of cell distortion in area of squa
mous metaplasia cja^be easily determined, x 770.
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Fig. 12. Trachéalepithelium 1 week after 10 BP plus ferric oxide instillations. Distorted squamous epithelial cell from Fig. 11. Note surface charac
ter of underlying cells, x 2310.
Fig. 13. Clump of ferric oxide particles, identified by X-ray diffraction, x 2000.
Fig. 14. Trachéalepithelium 4 weeks after 10 instillations of BP plus ferric oxide. Numerous holes (arrows) mark the former location of ciliated
cells. Note ferric oxide particle (F) and while blood cells (W), x 770.
Fig. 15. Trachéal
epithelium I week after 10 instillations of BP plus ferric oxide. Large groups of cells protruding into trachea! lumen, x 610.
Fig. 16. Trachea! epithelium 4 weeks after last BP plus ferric oxide instillations. Single enlarged, protruding nonciliated cell with protruding surface.
x 3500.
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Port, Henry, Kaufman,
Harris, and Ketels
Fig. 17. Trachea! epithelium 4 weeks after last BP plus ferric oxide instillations. Cluster of enlarged nonciliated cells, x 700. A, x 2310 (A, inset, is
further magnified at upper right, x 7700): B, x 3850; C, x 7700.
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VOL. 33
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Acute Changes in the Surface Morphology of Hamster
Tracheobronchial Epithelium following Benzo( a)pyrene and
Ferric Oxide Administration
Curtis D. Port, Mary C. Henry, David G. Kaufman, et al.
Cancer Res 1973;33:2498-2506.
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