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Ultrastructure of pulmonary adenomas induced by intratracheal instillation of diethylnitrosamine in Syrian golden hamsters
Europ. d. Cancer Vol. 9, pp. 359-362. Pergamon Press 1973. Printed in Great Britain
Ultrastructure of Pulmonary Adenomas Induced by Intratracheal Instillation of Diethylnitrosamine in Syrian Golden Hamsters* W. STRAKS t and V. J. FERON~ tDepartment of Pathology and Centerfor Electron Microscopy, Medical Faculty, State University, Utrecht, The Netherlands ~Central Institute for Nutrition and Food Research TNO, Zeist, The Netherlands Abstra©t--Lung tumours induced in Syrian golden hamsters by multiple intratracheal instillations of diethylnitrosamine (DENA) were studied with semithin and electron microscopic techniques. Two distinctive types of epithelial cells could be identified, (a) small, cytologically.fairly undifferentiated cells and (b) larger well-differentiated cells closely resembling normal type II pneumocytes. No squamous epithelium wasfound in the turnouts.
INTRODUCTION
peripheral neoplasms were classified as benign growths consisting of adenomatous structures lined by stratified epithelium resembling squamous epithelium upon light microscopy. In an attempt to determine the type and orgin of this neoplastic epithelium, a few of such DENAinduced lung tumours were examined by electron microscopy. The present paper describes this ultrastructural study.
DIETI-IYLNITROSAMINE (DENA) has repeatedly been shown to be carcinogenic for the respiratory system of Syrian golden hamsters [1-9]. The neoplastic response in the nasal cavity, larynx, and trachea was generally found to be much higher than that in the lower respiratory tract [3, 4, 7-11]. A high incidence of lower respiratory tract tumours; however, was observed in hamsters when subcutaneous injection of DENA was followed by intratracheal instillation of ferric oxide [8, 10] which has never been found to possess carcinogenic activity of its own despite extensive testing [10, 12]. Moreover, Feron et al. [13] have recently shown that repeated intratracheal instillations of relatively high doses of DENA alone and with ferric oxide resulted in considerable numbers of tumours localized in the bronchiolo-alveolar region of Syrian golden hamsters. Nearly all of these
MATERIAL AND METHODS
Tumour material was obtained from 1 male and 1 female Syrian golden hamster which had received intratracheal instillations of 0-1 ml 0.5°//0 diethylnitrosamine (EGA-Chemie K.G., Keppler and Reif, Steinheim/Albuch, WGermany) in 0"9% NaC1 (sterile, non-pyrogenic, Baxter Laboratories, A. Christiaens N.V. Brussels, Belgium) once every 2 weeks for a period of 1 yr starting at an age of 11 weeks. Both hamsters were 75 weeks old when they were killed by intra-abdominal injection of Brietal sodium (Lilly Co., Indianapolis, Ind., U.S.A.). The origin of the animals, their housing and feeding were the same as described previously [ 13]. Immediately after death samples of several
Accepted 2 February 1973. *This work was supported in part by a grant from the research fund of the Scientific Advisory Committee Smoking and Health. This fund has been established by the Dutch Cigarette Industry Foundation. Reprint requests to V . J . Feron.
359
360
W. Straks and V. J. Feron
pulmonary tumours were cut into small tissue blocks of about 1 mm 3 in volume and immersed for 2 hr in ice-cold 2% glutaraldehyde buffered to pH 7.4 with 0"2 M phosphate-sucrose buffer at 0°C. The specimens were postfixed in 1% OsO4, dehydrated in graded alcohols and embedded in Epon. One micron sections were stained with silvermethenamine and methylene blue, and examined by phase-contrast microscopy. Thin sections (500 A) were stained with uranylacetate and lead citrate [14], and viewed with a Siemens Elmiskop I electron microscope at 60 kV.
cell types were lacking, but desmosomes did occur (Fig. 4). The larger cells also showed short microvilli at their luminal surface (Fig. 5). Many lysosomal structures and a few myelin figures were noticed in their cytoplasm. Moreover, these cells contained electron dense lamellar bodies, often showing lamellae nicely running parallel (Figs. 4, 5). These lamcllar bodies, which are highly characteristic for type II pneumocytes, occasionally showed signs of disintegration. The endoplasmic reticulum was slightly widened. Other cytoplasmic organelles were unremarkable.
RESULTS
DISCUSSION
Each of the two hamsters examined showed 2 pulmonary tumours measuring 0"2 to 0.7 cm in diameter. In addition, multiple tracheal papillomas were found in both animals. Gross and light microscopic appearance of both the pulmonary and tracheal neoplasms were the same as described previously [3, 4, 7-10, 13].
The lung tumours induced in hamsters by intratracheal instillations of DENA have been found, by light microscopy, to consist of adenomatous structures lined by stratified epithelium resembling squamous epithelium [13]. The present ultrastructural study, however, revealed no squamous epithelium in this type of pulmonary tumours. Even semi-thin plastic sections examined by phase-contrast microscopy did not provide sufficient detail to establish the absence of squamous cells, which demonstrates that electron microscopic techniques should be used if cell types in lung tumours are to be identified correctly. Two distinctive types of epithelial cells could be recognized: (a) small polygonal cytologically undifferentiated cells and (b) larger welldifferentiated cells resembling normal type II pneumocytes. By far the greatest part of the tumour cells were of the first type. These littledifferentiated cells, which were relatively deftcient in cytoplasmic organdies except for mitochondria, might have originated from type I pneumocytes. On the other hand, the presence of numerous desmosomes and complex interdigitations between the cells might indicate that the smaller tumour cells arose from basal calls of the trax:heobronchial epithelium and retained some of their ultrastructurai characteristics. The tracheal epithelium is deliberately mentioned as a possible site of origin, because the occurrence of the lung tumours could be due to transplantation of the tracheal papillary tumours into the lower respiratory tract as a consequence of the instillation procedure [13]. This type of DENA-induced tracheal neoplasms has often been described as squamons cell tumours [1-9]. However, a recent study of these tumours performed with semi-thin techniques revealed no metaplastic alterations to cornified or uncornified squamous cells [15]. Consequently, the absence of squamous epi-
Phase.contrast microscopy The pulmonary tumours consisted of fairly solid masses of connected cells often arranged in sheets around spaces indicating an adenomatous growth pattern (Fig. 1). The majority of the tumour cells was uniform in size and shape and contained a relatively large nucleus, whereas the cytoplasm was scanty. Connections seen between the cells resembled intercellular bridges (Fig. 2). Scattered among this uniform type of cells, larger cells containing relatively small nuclei and a considerable amount of cytoplasm appeared to be present mainly at the luminal border of the adenomatous structures (Figs. 1, 2). The abundant vesicular cytoplasm of these larger cells contained granular structures, the nature of which was obscure under the light microscope. Bronchiolar elements could not be discerned. Electron microscopy The smaller type of ceils contained many normal-appearing mitochondria, some lysosomes, but otherwise few cytoplasmic organelles. These cells were joined together by complex cytoplasmic interdigitations. There were numerous desmosomes, but no tonofilaxnents were observed. The free surface of the calls lining a lumen was provided with rather short, blunt microviUi which were located at irregular distances from each other (Fig. 3). The larger type of cells contained less electron dense cytoplasm than did the smaller cells. Cytoplasmic interdigitations between the two
Fig. 1.
Adenomatouspart of pulmonary tumour. A few small groups of relatively large cells containing vesicular cytoplasm occur among sheets of smaller dark cells (phase-contrast picture, silvermethenamine, × 800).
Fig. 2. Detail of pulmonary tumour. To the left 3 large cells containing abundant cytoplasm and to the right smaller cells containing scanty cytoplasm. Gonnections seen between the smaller cells are suggestive of intercellular bridges (phase-contrast picture, silvermethenamine, × 2000).
(to face p. 360)
Fig. 3. Small type of tumour cell. It has a prominent nucleus and only a small amount of cytoplasm, which contains several mitochondria (M). Complex cytoplasmic interdigitations and many desmosomes (arrows) characterize the intercellular relationship between these cells (uranyl acetate and lead citrate, x 13800).
Fig. 4. Tumour cells resembling normal type H pneumocytes. The cytoplasm contains many lysosomal structures and lamellar bodies (LM). The cells appear to be held together by desmosomes (arrows) and straight lengths of closely apposed cell membranes (uranyl acetate and lead citrate, x 15900).
Fig. 5. Tumour cells resembling type H pneumocytes. Note the short blunt microvilli at the free surface, lamellar bodies ( L M ) , desmosomes (arrows) and lysosomes (uranyl acetate and lead citrate, x 26500).
Ultrastructure of D E N A Induced Lung Adenomas in Hamsters thelium in the present lung tumours does not exclude their origin t~om tracheal neoplasms. The second type of cells occurring in the lung tumours closely resembled type II pneumocytes, though they did not always rest on a basal membrane. O n the other hand, they did form junctions with each other and with the smaller type of cells. It is not clear whether the larger cells are an essential part of the t u m o u r or represent a cellular (alveolar cell) reaction to the presence in the lung tissue of the true neoplastic cells, viz.. the small undifferentiated cells. T h e latter possibility seems to be preferable, since hyperpla, da of type II pneumocytes is known to occur under various pathological conditions [16-18]. In addition, great alveolar cell tumours reported so far in several animal species [19-23] and in m a n [24-26] indeed appeared to consist only of well-recognizable
type II pneumocytes invariably attached to a basal lamina. Further discussion on the cellular composition and the histogenesis of the tumours described here would lead to rather useless speculations. For the present, it is justified to state that the turnouts do not contain squamons epithelium but are comprised of fairly undifferentiated epithelial cells and larger cells closely resembling type II pneumocytes. Several experiments are currently being performed to obtain more information on the ultrastructural and biological characteristics of both the DENAinduced pulmonary and tracheal neoplasms in Syrian golden hamsters. A c l m o w l e d g e m e n t - - T h a n k s are due to Prof. Dr. T. Vossenaar and Dr. A. P. de Groot for helpful discussions and critically reviewing the manuscript.
RR~CF_~
1. 2. 3. 4. 5. 6.
7. 8. 9. 10.
11. 12.
13.
361
W. DONTENWlLLand U. MOHR,Tracheal- und Bronchialcarcinome bei Goldhamstern nach Behandlung mit Di~ithylnitrosamin. Klin. Wschr. 39, 493 (1961). W. DONTENWILL and U. MOHR, Carcinome des Respirationstractus nach Behandlung yon Goldhamstern mit Ditithylnitrosamin. Z. Krebsforsch. 64~ 305 (1961). K. ~¢[. HERROLDand L.J. DUNHAM,Induction of tumors in the Syrian hamster with diethylnitrosamine (N-nitrosodiethylamine). Cancer Res. 23, 773 (1963). K . M . H~RROLV, Epithelial papillomas of the nasal cavity: experimental induction in Syrian hamsters. Arch. Path. 78, 189 (1964). U. Monna, O. WmS~-Rand K. I~SLSTICr~R,Die Minimaldosis fiir die Wirkung yon Di~ithylnitrosamin aufdie Trachea beim Goldhamster. Naturwissenschaflen 53, 229 (1966). U. MORR, Effect of diethylnitrosamine in the respiratory system of Syrian golden hamsters. In: Morphology of Experimental Respiratory Car~'ncgencsis, Atomic Energy Commission Symposium Series 21 (ed. P. N~T'mSH~tM, M. G. HAVANAand J. W. D~.ATH~aAOP) p. 255, Oak Ridge, Tennessee, U.S. Atomic Energy Comm. Div. Techn. Inform. Extension (1970). R. MOm'mANO and U. SAt~Fma'rI, Carcinogenic response of the respiratory tract of Syrian golden hamsters to different doses of diethylnitrosamine. Cam'erRes. 28, 2197 (1968). R. Mom'Es~o, Systemic carcinogens (N-nitroso compounds) and synergistic or additive effects in respiratory carcinogenesis. Tumori 56, 335 (1970). R. MotwrmAa~oand U. SAFFma'rX,Carcinogenic response of the hamster respiratory tract to single subcutaneous administrations of diethylnitrosamine at birth. J. nat. GancerInst. 44~ 413 (1970). R. MO~SANO, U. SxtwtOT'rI and P. SHUBXK,The role of topical and systemic factors in experimental respiratory carcinogenesis. In: Inhalation Carcinogenesis, Atomic Energy Commission Symposium Series 18 (ed. M. G. HANNA, P. N~a'rESHEIM and J. R. GILB~.RT) p. 353, Oak Ridge, Tennessee, U.S. Atomic Energy Comm. Div. Teehn. Inform. Extension (1970). P. N~TT~SHmM, Respiratory carcinogenesis studies with the Syrian golden hamster: a review. Progr. exp. TumorRes. (Basel) 16, 185 (1972). U.S.~a~Fioa'ri, F. C~.FIsand P. Smmm, Histopathology and histogenesis of lung cancer induced in hamsters by carcinogens carried by dust particles. In: Lung Tumours in Animals (ed. L. S~.vEm). Proc. 3rd Quadrennial Int. Conf. Cancer, p. 537, Perugia (1966). V.J. FERON, P. EMm~LOTand T. VOSS~,~R, Lower respiratory tract tumours in Syrian golden hamsters after intratracheal instillations of diethylnitrosamine alone and with ferric oxide. EuroiO.J. Cancer8, 445 (1972).
362
W. Straks and V. J. Feron
E . S . REYNOLDS,The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208 (1963). 15. J. ALTHOFF,R. WXLSONand U. MoaR, Diethylnitrosamine-induced alterations in the tracheobronchial system of Syrian golden hamsters. J. nat. Cancer Inst. 46, 1067 (1971). 16. A.G. H•PPLESTON, N. A. WRIOHT and J. A. STEWART,Experimental alveolar lipoproteinosis following the inhalation of silica. J. Path. 101, 293 (1970). 17. R.E. BROOKS,Ultrastructure of lung lesions produced by ingested chemicals-I. Effect of the herbicide Paraquat on mouse lung. Lab. Invest. 25, 536 (1971). 18. V . E . GOULD, R. TOSCO, R. F. WHEELIS,N. S. GOULD and Y. KAPANCI, Oxygen pneumonitis in man. Ultrastructural observations on the development of alveolar lesions. Lab. Invest. 269 499 (I 972). 19. S. HATTORI,~V~.MATSUDAand A. WADA, An electron microscopic study of pulmonary adenomas in mice. Gann 56~ 275 (1965). 90. R . E . BROOKS,Pulmonary adenoma of strain A mice: an electron microscopic study, jr. nat. CancerInst. 41~ 719 (1968). 2 I. R . E . BROOKS,Ultrastructure of mouse pulmonary adenomas. In: Morphology of Experimental Respiratory Carcinogenesis, Atomic Energy Commission Symposium Series 21 (ed. P. NETTESHEIM,M. G. HANNAandJ. W. DEATm~OE) p. 185, Oak Ridge, Tennessee, U.S. Atomic Energy Comm. Div. Techn. Inform. Extension (I 970). 22. E. BUCCIARELLI,Trapianti isogenici in ospiti adulti e neonati di tumori polmonari indotti con idrazina solfato in topi BALB/c/Cb/Se: studio ultrastruturale. Lay. Anat. Pat. Perugia 31~ 19 (1971). 23. K. PER~:, I. HOD and T. A. NOBEL, Pulmonary adenomatosis of sheep (Jaagsiekte)--I. Ultrastructure of the tumor, or. nat. CancerInst. 46, 525 ( 1971). 24. J . S . ADAMSON,R. M. SE~OR and T. M.gRRILL, Alveolar cell carcinoma. An electron microscopic study. Amer. Rev. resp. Dis. 10@~550 (1969). 25. J . J . COALSON,J. A. MOHR, J. K. PmTLE, A. L. DF.~. and E. R. RHO.~mES, Electron microscopy of neoplasms in the lung with special emphasis on the alveolar cell carcinoma. Amer. Rev. resp. Dis. 101~ i81 (1970). 26. G. NASH, P. C. LANOLINAISand K. A. Gm~ENAWALD,Alveolar cell carcinoma: does it exist? Cancer29~ 322 (1972). 14.
Ultrastructure of Pulmonary Adenomas Induced by Intratracheal Instillation of Diethylnitrosamine in Syrian Golden Hamsters* W. STRAKS t and V. J. FERON~ tDepartment of Pathology and Centerfor Electron Microscopy, Medical Faculty, State University, Utrecht, The Netherlands ~Central Institute for Nutrition and Food Research TNO, Zeist, The Netherlands Abstra©t--Lung tumours induced in Syrian golden hamsters by multiple intratracheal instillations of diethylnitrosamine (DENA) were studied with semithin and electron microscopic techniques. Two distinctive types of epithelial cells could be identified, (a) small, cytologically.fairly undifferentiated cells and (b) larger well-differentiated cells closely resembling normal type II pneumocytes. No squamous epithelium wasfound in the turnouts.
INTRODUCTION
peripheral neoplasms were classified as benign growths consisting of adenomatous structures lined by stratified epithelium resembling squamous epithelium upon light microscopy. In an attempt to determine the type and orgin of this neoplastic epithelium, a few of such DENAinduced lung tumours were examined by electron microscopy. The present paper describes this ultrastructural study.
DIETI-IYLNITROSAMINE (DENA) has repeatedly been shown to be carcinogenic for the respiratory system of Syrian golden hamsters [1-9]. The neoplastic response in the nasal cavity, larynx, and trachea was generally found to be much higher than that in the lower respiratory tract [3, 4, 7-11]. A high incidence of lower respiratory tract tumours; however, was observed in hamsters when subcutaneous injection of DENA was followed by intratracheal instillation of ferric oxide [8, 10] which has never been found to possess carcinogenic activity of its own despite extensive testing [10, 12]. Moreover, Feron et al. [13] have recently shown that repeated intratracheal instillations of relatively high doses of DENA alone and with ferric oxide resulted in considerable numbers of tumours localized in the bronchiolo-alveolar region of Syrian golden hamsters. Nearly all of these
MATERIAL AND METHODS
Tumour material was obtained from 1 male and 1 female Syrian golden hamster which had received intratracheal instillations of 0-1 ml 0.5°//0 diethylnitrosamine (EGA-Chemie K.G., Keppler and Reif, Steinheim/Albuch, WGermany) in 0"9% NaC1 (sterile, non-pyrogenic, Baxter Laboratories, A. Christiaens N.V. Brussels, Belgium) once every 2 weeks for a period of 1 yr starting at an age of 11 weeks. Both hamsters were 75 weeks old when they were killed by intra-abdominal injection of Brietal sodium (Lilly Co., Indianapolis, Ind., U.S.A.). The origin of the animals, their housing and feeding were the same as described previously [ 13]. Immediately after death samples of several
Accepted 2 February 1973. *This work was supported in part by a grant from the research fund of the Scientific Advisory Committee Smoking and Health. This fund has been established by the Dutch Cigarette Industry Foundation. Reprint requests to V . J . Feron.
359
360
W. Straks and V. J. Feron
pulmonary tumours were cut into small tissue blocks of about 1 mm 3 in volume and immersed for 2 hr in ice-cold 2% glutaraldehyde buffered to pH 7.4 with 0"2 M phosphate-sucrose buffer at 0°C. The specimens were postfixed in 1% OsO4, dehydrated in graded alcohols and embedded in Epon. One micron sections were stained with silvermethenamine and methylene blue, and examined by phase-contrast microscopy. Thin sections (500 A) were stained with uranylacetate and lead citrate [14], and viewed with a Siemens Elmiskop I electron microscope at 60 kV.
cell types were lacking, but desmosomes did occur (Fig. 4). The larger cells also showed short microvilli at their luminal surface (Fig. 5). Many lysosomal structures and a few myelin figures were noticed in their cytoplasm. Moreover, these cells contained electron dense lamellar bodies, often showing lamellae nicely running parallel (Figs. 4, 5). These lamcllar bodies, which are highly characteristic for type II pneumocytes, occasionally showed signs of disintegration. The endoplasmic reticulum was slightly widened. Other cytoplasmic organelles were unremarkable.
RESULTS
DISCUSSION
Each of the two hamsters examined showed 2 pulmonary tumours measuring 0"2 to 0.7 cm in diameter. In addition, multiple tracheal papillomas were found in both animals. Gross and light microscopic appearance of both the pulmonary and tracheal neoplasms were the same as described previously [3, 4, 7-10, 13].
The lung tumours induced in hamsters by intratracheal instillations of DENA have been found, by light microscopy, to consist of adenomatous structures lined by stratified epithelium resembling squamous epithelium [13]. The present ultrastructural study, however, revealed no squamous epithelium in this type of pulmonary tumours. Even semi-thin plastic sections examined by phase-contrast microscopy did not provide sufficient detail to establish the absence of squamous cells, which demonstrates that electron microscopic techniques should be used if cell types in lung tumours are to be identified correctly. Two distinctive types of epithelial cells could be recognized: (a) small polygonal cytologically undifferentiated cells and (b) larger welldifferentiated cells resembling normal type II pneumocytes. By far the greatest part of the tumour cells were of the first type. These littledifferentiated cells, which were relatively deftcient in cytoplasmic organdies except for mitochondria, might have originated from type I pneumocytes. On the other hand, the presence of numerous desmosomes and complex interdigitations between the cells might indicate that the smaller tumour cells arose from basal calls of the trax:heobronchial epithelium and retained some of their ultrastructurai characteristics. The tracheal epithelium is deliberately mentioned as a possible site of origin, because the occurrence of the lung tumours could be due to transplantation of the tracheal papillary tumours into the lower respiratory tract as a consequence of the instillation procedure [13]. This type of DENA-induced tracheal neoplasms has often been described as squamons cell tumours [1-9]. However, a recent study of these tumours performed with semi-thin techniques revealed no metaplastic alterations to cornified or uncornified squamous cells [15]. Consequently, the absence of squamous epi-
Phase.contrast microscopy The pulmonary tumours consisted of fairly solid masses of connected cells often arranged in sheets around spaces indicating an adenomatous growth pattern (Fig. 1). The majority of the tumour cells was uniform in size and shape and contained a relatively large nucleus, whereas the cytoplasm was scanty. Connections seen between the cells resembled intercellular bridges (Fig. 2). Scattered among this uniform type of cells, larger cells containing relatively small nuclei and a considerable amount of cytoplasm appeared to be present mainly at the luminal border of the adenomatous structures (Figs. 1, 2). The abundant vesicular cytoplasm of these larger cells contained granular structures, the nature of which was obscure under the light microscope. Bronchiolar elements could not be discerned. Electron microscopy The smaller type of ceils contained many normal-appearing mitochondria, some lysosomes, but otherwise few cytoplasmic organelles. These cells were joined together by complex cytoplasmic interdigitations. There were numerous desmosomes, but no tonofilaxnents were observed. The free surface of the calls lining a lumen was provided with rather short, blunt microviUi which were located at irregular distances from each other (Fig. 3). The larger type of cells contained less electron dense cytoplasm than did the smaller cells. Cytoplasmic interdigitations between the two
Fig. 1.
Adenomatouspart of pulmonary tumour. A few small groups of relatively large cells containing vesicular cytoplasm occur among sheets of smaller dark cells (phase-contrast picture, silvermethenamine, × 800).
Fig. 2. Detail of pulmonary tumour. To the left 3 large cells containing abundant cytoplasm and to the right smaller cells containing scanty cytoplasm. Gonnections seen between the smaller cells are suggestive of intercellular bridges (phase-contrast picture, silvermethenamine, × 2000).
(to face p. 360)
Fig. 3. Small type of tumour cell. It has a prominent nucleus and only a small amount of cytoplasm, which contains several mitochondria (M). Complex cytoplasmic interdigitations and many desmosomes (arrows) characterize the intercellular relationship between these cells (uranyl acetate and lead citrate, x 13800).
Fig. 4. Tumour cells resembling normal type H pneumocytes. The cytoplasm contains many lysosomal structures and lamellar bodies (LM). The cells appear to be held together by desmosomes (arrows) and straight lengths of closely apposed cell membranes (uranyl acetate and lead citrate, x 15900).
Fig. 5. Tumour cells resembling type H pneumocytes. Note the short blunt microvilli at the free surface, lamellar bodies ( L M ) , desmosomes (arrows) and lysosomes (uranyl acetate and lead citrate, x 26500).
Ultrastructure of D E N A Induced Lung Adenomas in Hamsters thelium in the present lung tumours does not exclude their origin t~om tracheal neoplasms. The second type of cells occurring in the lung tumours closely resembled type II pneumocytes, though they did not always rest on a basal membrane. O n the other hand, they did form junctions with each other and with the smaller type of cells. It is not clear whether the larger cells are an essential part of the t u m o u r or represent a cellular (alveolar cell) reaction to the presence in the lung tissue of the true neoplastic cells, viz.. the small undifferentiated cells. T h e latter possibility seems to be preferable, since hyperpla, da of type II pneumocytes is known to occur under various pathological conditions [16-18]. In addition, great alveolar cell tumours reported so far in several animal species [19-23] and in m a n [24-26] indeed appeared to consist only of well-recognizable
type II pneumocytes invariably attached to a basal lamina. Further discussion on the cellular composition and the histogenesis of the tumours described here would lead to rather useless speculations. For the present, it is justified to state that the turnouts do not contain squamons epithelium but are comprised of fairly undifferentiated epithelial cells and larger cells closely resembling type II pneumocytes. Several experiments are currently being performed to obtain more information on the ultrastructural and biological characteristics of both the DENAinduced pulmonary and tracheal neoplasms in Syrian golden hamsters. A c l m o w l e d g e m e n t - - T h a n k s are due to Prof. Dr. T. Vossenaar and Dr. A. P. de Groot for helpful discussions and critically reviewing the manuscript.
RR~CF_~
1. 2. 3. 4. 5. 6.
7. 8. 9. 10.
11. 12.
13.
361
W. DONTENWlLLand U. MOHR,Tracheal- und Bronchialcarcinome bei Goldhamstern nach Behandlung mit Di~ithylnitrosamin. Klin. Wschr. 39, 493 (1961). W. DONTENWILL and U. MOHR, Carcinome des Respirationstractus nach Behandlung yon Goldhamstern mit Ditithylnitrosamin. Z. Krebsforsch. 64~ 305 (1961). K. ~¢[. HERROLDand L.J. DUNHAM,Induction of tumors in the Syrian hamster with diethylnitrosamine (N-nitrosodiethylamine). Cancer Res. 23, 773 (1963). K . M . H~RROLV, Epithelial papillomas of the nasal cavity: experimental induction in Syrian hamsters. Arch. Path. 78, 189 (1964). U. Monna, O. WmS~-Rand K. I~SLSTICr~R,Die Minimaldosis fiir die Wirkung yon Di~ithylnitrosamin aufdie Trachea beim Goldhamster. Naturwissenschaflen 53, 229 (1966). U. MORR, Effect of diethylnitrosamine in the respiratory system of Syrian golden hamsters. In: Morphology of Experimental Respiratory Car~'ncgencsis, Atomic Energy Commission Symposium Series 21 (ed. P. N~T'mSH~tM, M. G. HAVANAand J. W. D~.ATH~aAOP) p. 255, Oak Ridge, Tennessee, U.S. Atomic Energy Comm. Div. Techn. Inform. Extension (1970). R. MOm'mANO and U. SAt~Fma'rI, Carcinogenic response of the respiratory tract of Syrian golden hamsters to different doses of diethylnitrosamine. Cam'erRes. 28, 2197 (1968). R. Mom'Es~o, Systemic carcinogens (N-nitroso compounds) and synergistic or additive effects in respiratory carcinogenesis. Tumori 56, 335 (1970). R. MotwrmAa~oand U. SAFFma'rX,Carcinogenic response of the hamster respiratory tract to single subcutaneous administrations of diethylnitrosamine at birth. J. nat. GancerInst. 44~ 413 (1970). R. MO~SANO, U. SxtwtOT'rI and P. SHUBXK,The role of topical and systemic factors in experimental respiratory carcinogenesis. In: Inhalation Carcinogenesis, Atomic Energy Commission Symposium Series 18 (ed. M. G. HANNA, P. N~a'rESHEIM and J. R. GILB~.RT) p. 353, Oak Ridge, Tennessee, U.S. Atomic Energy Comm. Div. Teehn. Inform. Extension (1970). P. N~TT~SHmM, Respiratory carcinogenesis studies with the Syrian golden hamster: a review. Progr. exp. TumorRes. (Basel) 16, 185 (1972). U.S.~a~Fioa'ri, F. C~.FIsand P. Smmm, Histopathology and histogenesis of lung cancer induced in hamsters by carcinogens carried by dust particles. In: Lung Tumours in Animals (ed. L. S~.vEm). Proc. 3rd Quadrennial Int. Conf. Cancer, p. 537, Perugia (1966). V.J. FERON, P. EMm~LOTand T. VOSS~,~R, Lower respiratory tract tumours in Syrian golden hamsters after intratracheal instillations of diethylnitrosamine alone and with ferric oxide. EuroiO.J. Cancer8, 445 (1972).
362
W. Straks and V. J. Feron
E . S . REYNOLDS,The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208 (1963). 15. J. ALTHOFF,R. WXLSONand U. MoaR, Diethylnitrosamine-induced alterations in the tracheobronchial system of Syrian golden hamsters. J. nat. Cancer Inst. 46, 1067 (1971). 16. A.G. H•PPLESTON, N. A. WRIOHT and J. A. STEWART,Experimental alveolar lipoproteinosis following the inhalation of silica. J. Path. 101, 293 (1970). 17. R.E. BROOKS,Ultrastructure of lung lesions produced by ingested chemicals-I. Effect of the herbicide Paraquat on mouse lung. Lab. Invest. 25, 536 (1971). 18. V . E . GOULD, R. TOSCO, R. F. WHEELIS,N. S. GOULD and Y. KAPANCI, Oxygen pneumonitis in man. Ultrastructural observations on the development of alveolar lesions. Lab. Invest. 269 499 (I 972). 19. S. HATTORI,~V~.MATSUDAand A. WADA, An electron microscopic study of pulmonary adenomas in mice. Gann 56~ 275 (1965). 90. R . E . BROOKS,Pulmonary adenoma of strain A mice: an electron microscopic study, jr. nat. CancerInst. 41~ 719 (1968). 2 I. R . E . BROOKS,Ultrastructure of mouse pulmonary adenomas. In: Morphology of Experimental Respiratory Carcinogenesis, Atomic Energy Commission Symposium Series 21 (ed. P. NETTESHEIM,M. G. HANNAandJ. W. DEATm~OE) p. 185, Oak Ridge, Tennessee, U.S. Atomic Energy Comm. Div. Techn. Inform. Extension (I 970). 22. E. BUCCIARELLI,Trapianti isogenici in ospiti adulti e neonati di tumori polmonari indotti con idrazina solfato in topi BALB/c/Cb/Se: studio ultrastruturale. Lay. Anat. Pat. Perugia 31~ 19 (1971). 23. K. PER~:, I. HOD and T. A. NOBEL, Pulmonary adenomatosis of sheep (Jaagsiekte)--I. Ultrastructure of the tumor, or. nat. CancerInst. 46, 525 ( 1971). 24. J . S . ADAMSON,R. M. SE~OR and T. M.gRRILL, Alveolar cell carcinoma. An electron microscopic study. Amer. Rev. resp. Dis. 10@~550 (1969). 25. J . J . COALSON,J. A. MOHR, J. K. PmTLE, A. L. DF.~. and E. R. RHO.~mES, Electron microscopy of neoplasms in the lung with special emphasis on the alveolar cell carcinoma. Amer. Rev. resp. Dis. 101~ i81 (1970). 26. G. NASH, P. C. LANOLINAISand K. A. Gm~ENAWALD,Alveolar cell carcinoma: does it exist? Cancer29~ 322 (1972). 14.