LCANCER
RESEARCH
36, 2659-2664.
July1976]
Fate of Early Carcinogen-induced Lesions in Tracheal
Epithelium'
Richard A. Griesemer,2 Paul Nettesheim, and Ann C. Marchok
Cancer and Toxicology Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
Summary
Hyperplastic-metaplastic lesions were produced in trans
planted rat tracheas by exposure to 310 or 75 j@g7,12dimethylbenz(a)anthracene or to 650 @zg
benzo(a)pyrene de
livened oven a 2-week exposure period. Tissue recovery was
studied for up to 16 weeks.
After cessation of carcinogen exposure, most of the hy
perplastic-metaplastic epithelium was rapidly replaced by
mucociliany epithelium . In the 7,12-dimethylbenz(a)anthna
cene-exposed tracheas, a few metaplastic foci remained at
4 and
8 weeks.
In all 3 exposure
groups,
portions
of the
mucosa were occupied by atnophic-pleomorphic epithelium
that persisted throughout the observation period. The sig
nificance of these lesions in the evolution of neoplasia is
as yet unknown.
In vitro studies with epithelial cells derived from 7,12dimethylbenz(a)anthracene-exposed
tracheal transplants
revealed that some cells have acquired a growth potential
that is markedly different from that of normal mucociliary
epithelium. This altered in vitro growth potential of epithe
hal cells may be an early indicator of an important event in
the evolution of epithelial transformation in vivo.
Introduction
A variety of epithelial changes have been described in the
respiratory tracts of humans inhaling tobacco smoke (4)
and of animals exposed to known carcinogenic agents (2,
3). There is reason to believe that some of these lesions are
t@ignificant links in the evolutionary chain of events leading
to the development of bronchogenic carcinoma. Those
changes that are considered to represent more severe cyto
logical and histological abnormalities (e.g. , metaplasia with
severe atypia) tend to occur more frequently at higher Cu
mulative exposures and in closer temporal relationship with
the appearance of bnonchogenic carcinoma (4). The pres
ently available information is inadequate, however, either to
distinguish cytotoxic effects from ‘carcinogenic'‘
effects
with any degree of certainty or to know the neoplastic
potential of any given epithelial lesion except invasive canci
I Presented at the Conference Early Lesions and the Development of
Epithelial Cancer,―October 21 to 23. 1975, Bethesda. Md. Research jointly
supported by the Carcinogenesis Program of the National dancer Institute,
Environmental Protection Agency. and the U. S. Energy Research and Devel
opment Administration under contract with the Union Carbide Corporation.
2 Presenter.
noma. Such information is of paramount importance to
identify early stages in the evolution of respiratory tract
neoplasia and to permit therapeutic intervention before pro
gression to invasive neoplasia.
The basic features of human bronchogenic carcinoma
and lung cancer can be reproduced in several animal spe
dies. If experimental studies are to contribute significantly
to the final goal of diagnosing and eradicating preneoplas
tic lesions, however, we must learn to do more than to
reproduce the human disease in animals. Experimental
models are needed that allow induction of epithelial lesions
(precancerous lesions) in a reproducible fashion at a pre
dictable site with a predetermined carcinogen dose and a
known exposure duration. The 1st attempts approaching
these requirements were made by Andervont (1), Kuschnem
and Laskin (7), and Stanton and Blackwell (10). Recently, 2
new “localized
tumor induction systems―for the respiratory
tract were developed in our own laboratory (6, 9). One of
these, the tracheal-transplant model, was used in the pres
ent study to investigate the fate in vivo and the behavior in
vitro of respiratory tract epithelium exposed to known quan
tities of carcinogen and showing distinct morphological
abnormalities. The findings discussed are part of an ongo
ing study which will be presented in full upon completion.
Specifically, tracheal epithelium was exposed for 2 weeks to
different doses of DMBA3 and BaP, which induced hypen
plastic-metaplastic lesions in all samples. The carcinogen
exposure was terminated at that time, and the morphologi
cal changes following removal of carcinogen were moni
toned for up to 16 weeks. In a parallel study, tracheal mu
cosa was placed in vitro after cessation of carcinogen expo
sure and the growth characteristics of the epithelium were
studied.
Materials and Methods
Fischer 344 pneumonia-free rats were used. The methods
for transplanting tracheas and exposing them to carcino
gens dissolved in beeswax have been reported previously
(5). Tracheal transplants were exposed for 2 weeks to car
cinogen-containing pellets. The transplants were then in
cised and the carcinogen pellets were removed. During the
2-week exposure period, 310 or 75 @.tg
of DMBA on 650 @.tg
of
BaP were delivered to each trachea, as calculated from the
3 The abbreviations
used are: DMBA,
7,12-dimethylbenz(a)anthracene;
BaP, benzo(a)pyrene.
JULY 1976
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R. A. Griesemer et a!.
amount of carcinogen remaining in the pellets at the end of
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RICOVMIICxLIAR1.05
COLUMNC&JSF
HYPIFRAFSI
the exposure. At intervals of 0, 1, 2, 4, 8, and 16 weeks after
removal of the carcinogen pellets, groups of 6 to 12 tracheal
transplants were fixed in Bouin's fluid and processed for
histological evaluation. Tracheas were cut transversely into
2-mm-long cylinders, all of which were embedded on end in
paraffin. The extent of the lesions in the cross-sections was
then estimated with the use of an ocular grid.
For convenience in scoring, a descriptive histological
Chart 1. Semiquantitative evaluation of the histological lesions in tra
classification of epithelial changes was selected (Table 1). cheas exposed to 310 @gDMBA delivered over 2 weeks compared with those
permitted a 1- to 4-week recovery (RECOV). F, focal lesions affecting less
The terms are arbitrarily arranged from normal through
than 5% of the mucosal surface. One square represents the entire surface
hyperplasia to metaplasia. The term “transitional
hyperpla
area compiled from 6 to 12 tracheas. HYPER, hyperplasia.
sia―denotes stratified hypemplastic lesions without defini
tive differentiation. The terms “pleomorphic―
and “atrophic
40%oftheepitheliallining
consisted ofan undifferentiated,
epithelium― are descriptive of persistent tracheal lesions
pleomorphic epithelium, 1 to several cell layers thick (Fig.
that do not fit into conventional classifications and that
5). A small proportion of the epithelial surface showed an
show nuclear and cellular atypia without signs of diffemen
attenuated and flattened epithelium (Fig. 6B). Occasionally,
tiation, on markedly attenuated epithelium, respectively.
this atrophic epithelium consisted of a double row of homi
To obtain information about the growth potential of the
zontally arranged flattened cells. The significance and
early epithelial lesions produced by DMBA, tracheas col
eventual fate of these pleomomphic and atrophic lesions
lected at the end of the 2-week in vivo exposure period were
seen at 4 weeks of recovery are presently not known.
cut into small pieces and were cultured as explants de
In a parallel study, pieces of tracheas exposed in vivo to
signed to give epithelial outgmowths (8) under in vitro condi
the same DMBA dose for 2 weeks were cultured as explants
tions.
under defined conditions. The main purposes of these in
vivo-in vitro studies were to determine the frequency with
which epithelial outgrowths could be established from tra
Results
cheal explant cultures and the rate at which this outgrowth
of epithelial cells would occur. From control cultures not
Lesions Induced by 310 @zg
DMBA. In previousstudies
exposed to carcinogenic substances, ciliated epithelial out
with transplanted mat tracheas, we had established that
growths were regularly obtained in Waymouth's medium.
doses of 280 @g
DMBA and above delivered oven a 2-month
Under the same conditions, only a small proportion of the
period produced 100% invasive squamous cell carcinomas
explants established from the carcinogen-exposed tracheas
within that time, while 100 j.@gDMBA induced 50% invasive
yielded epithelial outgmowths. However, cell lines could be
lesions in 6 to 8 months. The high DMBA dose of 310 @g
was
established from some of the outgrowths derived from
selected in the present study because it is likely to be
DMBA-treated tracheas (Fig. 7). This suggests a marked
oncogenic even when delivered over a short time period
difference in growth potential between DMBA-exposed tra
(the tumor induction data from this study are not yet availa
cheal epithelium and normal tracheal epithelium. The cell
ble).
lines derived from DMBA-exposed tracheas are presently
The semiquantitative evaluation of epithelial changes in
being tested for oncogenic potential in animals.
tracheal transplants is summarized in Chart 1. At the end of
LesionsInducedby 75 @zg
DMBA. In previoustumonige
the 2-week exposure, the mucosal epithelium was uniformly
nesis studies, this dose of carcinogen produced a tumor
replaced by thick keratinizing squamous epithelium (Figs. 1
incidence of 50% or less. When delivered over 2 weeks, this
and 2). After a recovery period of 1 week, following the
dose produced generalized squamous metaplasia, similar
removal of carcinogen, there was a dramatic change of the
to that with the higher DMBA dose, although the mucosa
tracheal epithelium to transitional hyperplasia (Fig. 3), with
varied more in thickness. After removal of the carcinogen
only 20% squamous metaplasia remaining. After 4 weeks of
pellets, there was a rapid return of most of the mucosal
recovery, only a few tiny foci of squamous metaplasia per
epithelium to columnar, mucociliary cells (Chart 2), al
sisted. Nearly half ofthe mucosal surface was covered with
though small metaplastic foci persisted throughout the pe
near-normal columnar epithelium (Fig. 4A). Approximately
nod of observation. Small portions of tracheal epithelium
consisted of pleomorphic and atrophic epithelium at 2 to 8
weeks of recovery. Histological observations beyond 8
Table 1
weeks of recovery are not yet available, but palpation 32
Histological
classification
of tracheal epithelial lesions
weeks after removal of carcinogen reveals no tumors.
Histological classification
When explanted in vitro , the rate of epithelial outgrowth
1 . Mucociliary epithelium
from tracheas exposed to 75 @gDMBA within a 2-week
2. Low columnar epithelium
exposure period was more rapid than that from control
3. Columnar cell hyperplasia
explants. Again several cell lines could be established from
4. Transitional hyperplasia
5. Squamous metaplasia
such outgrowths (Fig. 8), whereas normal explants yielded
6. Pleomorphic epithelium
only short-term primary cultures. This indicates that even at
7. Atrophic epithelium
this low carcinogen dose, marked changes in epithelial
2660
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RESEARCH
VOL. 36
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Fate of Carcinogen-induced
Tracheal Lesions
[email protected]
FRYER@TAPLASIAPLEOIIOSPSCAIFOPsV0i@i@____=
COLUSUC8.APIU
RIPERTRMSfl
5(115 H@P@RM(TAPLASIAP@IQM@FPwm@AtROP1II0@;=_=2“7@7@
RECOYNUCOCItIARYiOSCOu*CO1@NYP1RTRAJISIT
221±===87@7@@?@:z=_-@--=z:z:
Chart 2. Evaluation of the histological lesions in tracheas exposed to 75
;Lg of DMBA compared with those permitted a 1- to 8-week recovery (RE
COy). F, focal lesions involving less than 5% of the mucosal surface. HYPER,
hyperplasia.
Chart 3. Evaluation of the histological lesions in tracheas exposed to 650
iLg of BaP compared
with
those
permitted
a 1- to 16-week
recovery
(RECOV).
Hyper, hyperplasia.
growth characteristics exist which distinguish normal from
carcinogen-exposed epithelium.
Lesions Inducedby 650 @g
BaP. Previousstudieshave
shown that BaP doses of 1000 to 1200 j@gdelivered over a 6month time period are tumonigenic in 100% of the animals,
with tumors appearing at about 8to 10 months. Whether the
dose applied in the present study is carcinogenic is not
known . After delivery within 2 weeks of 650 @g
of BaP to the
tracheal transplants, the tracheal lining was characterized
by transitional and hyperplastic columnar epithelium (Chart
3). Squamous metaplasias were not seen. A characteristic
feature not observed in the other 2 groups was the develop
ment of rather large erosions in most of the tracheal trans
plants. As in the DMBA-exposed tracheas, normal or near
normal mucociliary epithelium returned rapidly after me
moval of the carcinogen. Pleomorphic and atrophic epithe
hum appeared late in the recovery period, accompanied by
a few foci of persistent erosion. Most of the epithelium,
however, appeared normal at 16 weeks. No palpable tumors
have appeared in tracheas maintained for 32 weeks after
removal of carcinogen.
Discussion
The data reported here represent our initial findings in a
series of studies designed to elucidate the fate and neoplas
tic potential of carcinogen-induced lesions in respiratory
tract epithelium. The epithelium in which these lesions were
induced was exposed to a known quantity of carcinogen for
a known period of time. Two questions were asked in the
study. (a) Do the morphologically recognizable epithelial
lesions induced by a defined carcinogen dose regress after
removal of the carcinogen, do they persist, or do they give
way to a different epithelial lesion, and is there any indica
tion of “progression―
of one lesion to another? (b) Can we
detect significant changes in the growth behavior of epithe
hal cells in vitro following a limited carcinogen exposure
which may on may not be tumonigenic? The experiment was
facilitated by the fact that, at least with the 2 DMBA doses
chosen , the morphological change seen at 2 weeks of expo
sure (namely, squamous metaplasia) encompassed the en
tire epithelial surface of the tracheal transplant. With cessa
tion of the carcinogen exposure, most of the epithelial
JULY 1976
surface area returned to a near-normal or normal state of
differentiation, leaving only a few small foci of squamous
metaplasia behind. Thus, most of this early metaplastic
change can be said to have regressed. Besides the occa
sional persisting metaplastic focus, however, some patches
of pleomorphic-atnophic epithelium having no specific type
of differentiation appeared. Whether this type of mompho
logical change is of any importance in the evolution of later
appearing lesions remains to be seen. Obviously, important
information is still lacking but should come from later time
points in the study. Although the morphological findings
suggest that most of the initial changes might be of little
relevance for the process of cancinogenesis, the in vitro
study suggests that at least some of the epithelial cells
emerging after this rather brief carcinogen exposure appear
to have acquired a rather strikingly different growth poten
tial. It is tempting to speculate that this altered in vitro
growth potential might be an early indicator of an important
event in the evolution of the transformed epithelial cells.
It is realized
that many questions
must be answered
be
fore these preliminary findings can be related to the evolu
tion of epithelial cancer in vivo, if indeed they are related.
However, we feel that our findings are promising and that
they demonstrate the usefulness of the experimental ap
proach described in this paper in identifying early neoplas
ticchanges.
References
1. Andervont,
2.
3.
4.
5.
6.
H. B. Pulmonary
Tumors
in Mice. IV. Lung Tumors
Induced
by Subcutaneous Injection of 1,2,5,6-Dibenzanthracene in Different Me
dia and by Its Direct Contact with Lung Tissue. Public Health Rept. , 52:
1584-1589,1937.
Auerbach, 0. , Hammond, E. C. , Kirman, D. , and Garfinkel. L. Effects of
Cigarette Smoking on Dogs. II. Pulmonary Neoplasms. Arch. Environ.
Health, 21: 754-768, 1970.
Auerbach, 0. , Hammond, E. C., Kirman, D., Garfinkel, L., and Stout. A.
P. Histological Changes in Bronchial Tubes of Cigarette-smoking Dogs.
dancer, 20: 2055-2066, 1967.
Auerbach, 0., Stout, A. P., Hammond, E. C., and Garfinkel, L. Changes
in Bronchial Epithelium in Relation to Cigarette Smoking and in Relation
to Lung Cancer. New EngI. J. Med., 265: 253-267, 1961.
Gniesemer, R. A., Kendnick, J., and Nettesheim, P. Tracheal Grafts. In: E.
Karbe and J. F. Parks (eds.), Experimental Lung Cancer, Carcinogene
sis, and Bioassays, pp. 539-547. Berlin: Springer-Verlag. 1974.
Kendnick, J. , Nettesheim, P., and Hammons, A. S. Tumor Induction in
Tracheal Grafts: A New Experimental Model for Respiratory Carcinogen
2661
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R. A. Griesemer et a!.
esis Studies. J. NatI. Cancer Inst., 52: 1317-1326, 1974.
7. Kuschner, M. , and Laskin, S. Experimental Models in Environmental
Carcinogenesis. Am. J. Pathol., 64: 183-196, 1971.
8. Marchok, A. C. , Cone, v., and Nettesheim, P. Induction of Squamous
Metaplasia (Vitamin A Deficiency) and Hypersecretory Activity in Tra
cheal Organ Cultures. Lab. Invest., 33: 451-460, 1975.
9. Schreiber, H., Schreiber, K., and Martin, D. H. Experimental Tumor
Fig. 1 . Exposure of the trachea to 310 or 75
Induction in a Circumscribed Region of the Hamster Trachea: dorrela
tion of Histology and Exfoliative Cytology. J. NatI. Cancer Inst., 54: 187198, 1975.
10. Stanton, M. F., and Blackwell, R. Induction of Epidermoid Carcinoma in
Lungs of Rats: A “New―
Method Based upon Deposition of Methylchol
anthrene in Areas of Pulmonary Infarction. J. Natl. Cancer Inst.. 27: 375407,1961.
@g
of DMBA for 2 weeks resulted in squamous
metaplasia
ofthe entire tracheal mucosa. The lumen isfilled
with
keratin and cornified cells. DMBA, 310 @tg,
shown. H & E, x 100.
Fig. 2. Higher magnification of the metaplastic lining in Fig. 1. Submucosal glands are also affected. x 320.
Fig. 3. Transitional
hyperplasia
following
delivery of65O
@g
of BaP to the tracheal
mucosa. The mucosal lining is stratified
without
definitive
differentiation.
Similar lesions occurred in tracheas that had been exposed to DMBA and permitted 1 week recovery without additional carcinogen exposure. H & E, x 450.
Fig. 4. Four to 8 weeks after termination
of carcinogen
exposure,
the majority ofthe mucosal epithelium
in all 3 treatment
groupswas
simple mucociliary
in
type. but not yet normal. A, columnar and low-columnar epithelium after 310 @tg
of DMBA and 4 weeks of recovery. H & E, x 540. B, Columnar cell hyperplasia
after 650 lLg of BaP and 8 weeks of recovery. H & E, x 540.
Fig. 5. During the recovery period, small portions of the tracheal mucosa appeared pleomorphic and undifferentiated. A, abrupt transition from low
columnar (arrow)to pleomorphic epithelium. BaP, 650 @g,
and 2 weeks of recovery. H & E, x 560. B, thin pleomorphic epithelium. DMBA, 75 @g,
and 2 weeks
of recovery. H & E, x 560. C, stratified, hyperbasophilic epithelium with little differentiation. DMBA, 75 @g,
and 8 weeks of recovery. Note the disparity in cell
sizes. H & E, x 560.
Fig. 6. Other portions
of tracheal
mucosa were flattened
and atrophic.
A, 310 @zg
of DMBA and 1 week of recovery. H & E, x 560. B, 310 @igof DMBA and 4
weeks of recovery. H & E, x 560. C, 75 @g
of DMBA and 8 weeks of recovery. Note the abrupt transition from small, low-columnar cells. H & E, x 560.
Fig. 7. Cell line derived from the outgrowth of an explanted trachea that had been exposed to 310 @g
of DMBA. This cell line is characterized by small,
undifferentiated cells and rapid growth rate. Papanicolaou's stain. x 390.
Fig. 8. Cell line derived from the outgrowth of an explanted trachea that had been exposed to 75 @g
of DMBA. The epithelial cells of this line are much
larger than those in Fig. 7. Papanicolaou's stain, x 390.
2662
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RESEARCH
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VOL. 36
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2664
CANCER
RESEARCH
VOL. 36
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1976 American Association for Cancer Research.
Fate of Early Carcinogen-induced Lesions in Tracheal
Epithelium
Richard A. Griesemer, Paul Nettesheim and Ann C. Marchok
Cancer Res 1976;36:2659-2664.
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