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Pathogenesis of Diethylnitrosamine-Induced Tumors in the Trachea of the Syrian Golden Hamster
Path. Res. Pract. 168, 185-192 (1980)
NCI Frederick Cancer Research Center Frederick, MD 21701, USA
Pathogenesis of Diethylnitrosamine-Induced Tumors in the Trachea of the Syrian Golden Hamster* HILDEGARD M. REZNIK-SCHULLER
Summary The pathogenesis of tracheal tumors induced in Syrian hamsters by subcutaneous treatment of N-nitrosodiethylamine (DEN) was examined in a serial sacrifice study using light and electron microscopy. Initial ultrastructural changes were noted in ciliated and mucous cells, and basal cells were identified as source of the later developing papillary polyps and squamous carcinomas.
Introduction The pronounced organotropy of DEN for the respiratory tract of the Syrian hamster has been well documented (Dontenwill and Mohr, 1961; Dontenwill et aI., 1962; Herrold and Dunham, 1963, Herrold, 1964, Montesano and Saffioti, 1968). To study this phenomenon, it is important that the target cells be identified. This can be done by following the development of the induced neoplasms sequentially. Serial sacrifice studies in DEN-treated Syrian Golden hamsters have shown that the induced adenomatous lung tumors originate from bronchial Clara cells and APUD-type cells (Reznik-Schuller, 1976a, b; 1977). To extend these studies, the sequential development of DEN-induced neoplasms in the trachea has now been examined.
Materials and Methods Forty four male Syrian Golden hamsters were injected subcutaneously twice a week for life with 1110 the LDso (178 mglkg b.w.) of DEN. Ten control animals were similarly injected with the solvent alone (physiological saline). From the 2nd to the 20th week of treatment, two animals were sacrificed each week. Four of the dosed animals were allowed to survive until moribund (31,
* This work was supported by Contract No. NOI-CO-75380 with the National Cancer Institute, NIH, Bethesda, Maryland 20014.
186· Hildegard M. Reznik-Schuller 34, 35, 36 weeks after start of the experiment). All animals were fixed in situ by vascular perfusion with 2% cacodylate-buffered glutaraldehyde (pH 7.4). The tracheas were removed and cut into individual rings under a stereomicroscope. Three rings from each trachea (3rd, 6th, 12th ring) were processed for electron microscopy (postfixation in 1 % 0504; dehydration; embedding in Epon 812). Flat embedding was used to facilitate proper orientation of epithelia for cutting with a LKB ultrotome III. Semithin sections were stained with toluidine blue. Ultrathin sections were mounted on uncoated copper grids and stained with uranyl acetate and lead citrate. They were examined in a Philips 201C electron microscope, electron micrographs being taken at an accelerating voltage of 60 KV.
Results The epithelial composition and ultrastructure of cells in the tracheas of the control animals did not show any abnormalities, and displayed the same morphology as that recently described in detail by Becci et aI., (1978). Up to the 4th week of DEN-treatment, the cellular composition of the tracheal epithelia appeared normal. Electron microscopy revealed occasional ciliated cells with proliferated smooth endoplasmic reticulum, while rough endoplasmic reticulum was extensive in most mucous cell. Starting with the 3rd week of treatment, a progressing increase in the amount of lysosomal derivatives occurred in ciliated cells. As early as 5 weeks after DEN-treatment was initiated, occasional ciliated and mucous cells exhibited loss of polarity, the orientation of the nuclei changing from perpendicular to parallel to the basement membrane (Fig. 1).
Fig. 1. Epithelial cells after 5 weeks of DEN-treatment: the orientation of a nucleus has changed from a perpendicular orientation to one parallel to the basement membrane. x 6000.
Pathogenesis of Diethylnitrosamine-Induced Tumors' 187
This was followed by a proliferation of basal cells (Fig. 2), starting at week 6. During the following weeks, progressive epithelial hyperplasia developed, which was accompanied by gradual stratification of epithelia, basal cells form-
Fig. 2. Tracheal epithelium after 6 weeks of DEN-treatment: basal cells have started to proliferate. x 600.
Fig. 3. Middle layer of hyperplastic tracheal epithelium after 8 weeks of DEN-treatment: This area is occupied by cells which appear transitional in their differentiation between basal and mucous cells. x 12 000.
188 . Hildegard M. Reznik-Schuller
Fig. 4. Trachea in the 13th week of DEN-treatment: papillary polyp has developed, the basal part of which is shown in this picture. X 8000.
Fig. 5. Part of papillary polyp after 14 weeks of DEN-treatment: The cytoplasm of a mucous cell contains many mucous granules. x 6000.
Pathogenesis of Diethylnitrosamine-Induced Tumors· 189
Fig. 6. Squamous metaplasia in another part of the papillary polyp shown in Fig. 5: The cytoplasm contains thick bundles of filaments while other cell organelles are scanty. x 18300.
Fig. 7. Squamous carcinoma in the trachea after 34 weeks of DEN-treatment: the tumor cells have invaded the subepithelial tissue and cartilage. X 100.
190 . Hildegard M. Reznik-Schiiller
ing the basal layer. The middle layer of such hyperplastic areas was occupied by cells which seemed to be transitional in their differentiation between basaland mucous cells (Fig. 3). They did contain a well-developed endoplasmic reticulum, like mucous cells, but unlike the latter, did not show any production of mucous. The surface layer of the hyperplastic epithelia was composed of fully differentiated mucous cells while ciliated cells were scanty. The first tracheal tumors, papillary polyps (Fig. 4), were seen in the 13th week and were found in all of the animals sacrificed thereafter. The neoplasms consisted almost entirely of transitional and mucous cells, while basal cells were restricted to the basal layer and ciliated cells were lacking completely. The cytoplasm of some mucous cells was occupied by mucous granules (Fig. 5). Other mucous cells, as well as some of the transitional cells, exhibited various amonts of filament bundles in their cytoplasm, which indicates that these cells were undergoing squamous metaplasia (Fig. 6). Two of the four animals which survived more than 20 weeks had squamous carcinomas in addition. These neoplasms invaded the subepithelial tissue and cartilage (Fig. 7). At the ultrastructural level such tumor cells were characterised by abundant cytoplasmic filaments and sparseness of other cytoplasmic organelles. During the entire period of observation, no proliferation of APUD-type cells was seen in the tracheal epithelium.
Discussion Previous sequential studies of the development of DEN-induced pulmonary tumors in Syrian hamsters have revealed early ultrastructural changes in bronchial Clara cells and APUD-ceIls, and these two cell types were also the sources of the neoplasms (Reznik-SchUller, 1976a, b, 1977). The high content of oxidative enzymes in Clara cells (Azzopardi and Thurlbeck, 1969), together with the ability of APUD-cells to take-up and metabolize simple amines and their precursors (Pearse, 1974), lead us to the conclusion that metabolic competence is an important factor in the selective action of nitrosamines (ReznikSchUller and Lijinsky, 1979). In view of this, mucous cells would seem the most likely site for nitrosamine activation in the tracheal epithelium because they are metabolically the most active cells in this area (Azzopardi and Thurlbeck, 1968). It is surprising, therefore, that it was the metabolically most inactive epithelial cells (Kaufmann et al. 1972), the basal cells, that were the origin of neoplastic development in the trachea although these cells were not affected during the initial treatment period. When one searches for common characteristics of cell types which produce tumors in both bronchi and trachea, it becomes evident that bronchial Clara
Pathogenesis of Diethylnitrosamine-Induced Tumors· 191
cells and tracheal basal cells are both stem cells for cell renewal (Evans et aI., 1976; Shorter, Titus and Divertie, 1964). In the bronchus, one target cell of DEN is a stem cell, the Clara cell, which is metabolically very active, while in the trachea, the target cell is also a stem cell, the basal cell, which is metabolically inactive. This suggests that while metabolic competence is important, it is not the only factor. For cells to be capable of undergoing neoplastic change, a preexisting ability to proliferate seems important. It is also possible that the ultimate carcinogenic metabolite of the nitrosamine is formed in another cell type, in the immediate vicinity of the target Clara or basal cells (e.g. in mucous cell in the trachea). From our present knowledge, this is a rather theoretical consideration. We require further information on the intracellular site of binding and metabolism of the inducing carcinogen to settle this problem. Acknowledgements. The authors would like to thank Ms. Lee Ann Nutter for excellent technical assistance. This work was initiated at the section of Experimental Pathology of the Medical University, Hannover (FRG).
References Azzopardi, A., and Thurlbeck, W. M.: Oxidative enzyme pattern of the bronchial mucous glands. Amer. Rev. resp. Dis. 97, 1038-1045 (1968) Azzopardi, A., and Thurlbeck, W. M.: The histochemistry of the nonciliated bronchiolar epithelial cell. Amer. Rev. resp. Dis. 99, 516-525 (1969) Becci, P. J., McDowell, E. M., and Trump, B. F.: The respiratory epithelium. II. Hamster trachea, bronchus, and bronchioles. J. mat. Cancer Inst. 61, 551-562 (1978) Dontenwill, W., and Mohr, U.: Carcinome des Respirationstraktes nach Behandlung von Goldhamstern mit Diaethylnitrosamin. Z. Krebsforsch. 64, 305-312, (1961) Dontenwill, W., Mohr, U., and Zagel, M.: Uber die unterschiedliche Lungencarcinogene Wirkung von Diaethylnittosamin bei Hamster und Ratte. Z. Krebsforsch. 64, 499-502 (1962) Evans, M. J., Johnson, L. V., Stephens, R. J., and Freeman, G.: Renewal of the terminal bronchiolar epithelium in the rat following exposure to N0 2 or Os. Lab. Invest. 35, 246-257 (1976) Herrold, J. M.: Effect of route of administration on the carcinogenic action of diethylnitrosamine (N-nitroso-diethylamine). Brit. J. Cancer 18, 763-767 (1964) Herrold, K. M., and Dunham, L.: Induction of tumors in the Syrian Golden hamster with diethylnitrosamine (N-N-nitrosodiethylamine). Cancer Res. 23, 773-777 (1963) Kaufman, D. G., Baker, M. S., Harris, C. c., Smith, J. M., Boren, H., Sporn, M. B., and Saffiotti, U.: Co-ordinated biochemical and morphological examination of hamster tracheal epithelium. J. nat. Cancer Inst. 49, 783-792, (1972) Montesano, R., and Saffiotti, U.: Carcinogenic response of the respiratory tract of Syrian Golden hamsters to different doses of diethylnitrosamine. Cancer Res. 28, 2197-2210 (1968) Pearse, A. G. E.: The APUD cell concept and its implications in pathology. Path. Annu. 9, 27-41 (1974) Reznik-Schuller, H.: Proliferation of endocrine (APUD-type) cells during early N-diethylnitrosamine-induced lung carcinogenesis in hamsters. Cancer Lett. 1, 255-258 (1976a)
192 . Hildegard M. Reznik-SchUller Reznik-Schiiller, H.: Ultrastructural alterations of nonciliated cells after nitrosamine treatment and their significance for pulmonary carcinogenesis. Amer. J. Path. 85, 549-552 (1976b) Reznik-Schiiller, H.: Ultrastructural alterations of APUD-cells during nitrosamine-induced lung carcinogenesis. J. Path. 121, 79-82 (1977) Reznik-Schiiller, H., and Lijinsky, W.: In vivo autoradiography and nitrosoheptamethyleneimine carcinogenesis in hamsters. Cancer Res. 39, 72-74, (1979) Shorter, R. G., Titus, J. L., and Divertie, M. B.: Cytodynamics in the respiratory tract. Dis. Chest
46, 138-142 (1964)
Received July 2, 1979 . Accepted August 17, 1979
Key words: Diethylnitrosamine - Carcinogenesis - Hamster - Trachea Ultrastructure - Mucous cells
Dr. Hildegard M. Reznik-SchUller, NCI Frederick Cancer Research Center, Frederick, MD,
21701, USA
NCI Frederick Cancer Research Center Frederick, MD 21701, USA
Pathogenesis of Diethylnitrosamine-Induced Tumors in the Trachea of the Syrian Golden Hamster* HILDEGARD M. REZNIK-SCHULLER
Summary The pathogenesis of tracheal tumors induced in Syrian hamsters by subcutaneous treatment of N-nitrosodiethylamine (DEN) was examined in a serial sacrifice study using light and electron microscopy. Initial ultrastructural changes were noted in ciliated and mucous cells, and basal cells were identified as source of the later developing papillary polyps and squamous carcinomas.
Introduction The pronounced organotropy of DEN for the respiratory tract of the Syrian hamster has been well documented (Dontenwill and Mohr, 1961; Dontenwill et aI., 1962; Herrold and Dunham, 1963, Herrold, 1964, Montesano and Saffioti, 1968). To study this phenomenon, it is important that the target cells be identified. This can be done by following the development of the induced neoplasms sequentially. Serial sacrifice studies in DEN-treated Syrian Golden hamsters have shown that the induced adenomatous lung tumors originate from bronchial Clara cells and APUD-type cells (Reznik-Schuller, 1976a, b; 1977). To extend these studies, the sequential development of DEN-induced neoplasms in the trachea has now been examined.
Materials and Methods Forty four male Syrian Golden hamsters were injected subcutaneously twice a week for life with 1110 the LDso (178 mglkg b.w.) of DEN. Ten control animals were similarly injected with the solvent alone (physiological saline). From the 2nd to the 20th week of treatment, two animals were sacrificed each week. Four of the dosed animals were allowed to survive until moribund (31,
* This work was supported by Contract No. NOI-CO-75380 with the National Cancer Institute, NIH, Bethesda, Maryland 20014.
186· Hildegard M. Reznik-Schuller 34, 35, 36 weeks after start of the experiment). All animals were fixed in situ by vascular perfusion with 2% cacodylate-buffered glutaraldehyde (pH 7.4). The tracheas were removed and cut into individual rings under a stereomicroscope. Three rings from each trachea (3rd, 6th, 12th ring) were processed for electron microscopy (postfixation in 1 % 0504; dehydration; embedding in Epon 812). Flat embedding was used to facilitate proper orientation of epithelia for cutting with a LKB ultrotome III. Semithin sections were stained with toluidine blue. Ultrathin sections were mounted on uncoated copper grids and stained with uranyl acetate and lead citrate. They were examined in a Philips 201C electron microscope, electron micrographs being taken at an accelerating voltage of 60 KV.
Results The epithelial composition and ultrastructure of cells in the tracheas of the control animals did not show any abnormalities, and displayed the same morphology as that recently described in detail by Becci et aI., (1978). Up to the 4th week of DEN-treatment, the cellular composition of the tracheal epithelia appeared normal. Electron microscopy revealed occasional ciliated cells with proliferated smooth endoplasmic reticulum, while rough endoplasmic reticulum was extensive in most mucous cell. Starting with the 3rd week of treatment, a progressing increase in the amount of lysosomal derivatives occurred in ciliated cells. As early as 5 weeks after DEN-treatment was initiated, occasional ciliated and mucous cells exhibited loss of polarity, the orientation of the nuclei changing from perpendicular to parallel to the basement membrane (Fig. 1).
Fig. 1. Epithelial cells after 5 weeks of DEN-treatment: the orientation of a nucleus has changed from a perpendicular orientation to one parallel to the basement membrane. x 6000.
Pathogenesis of Diethylnitrosamine-Induced Tumors' 187
This was followed by a proliferation of basal cells (Fig. 2), starting at week 6. During the following weeks, progressive epithelial hyperplasia developed, which was accompanied by gradual stratification of epithelia, basal cells form-
Fig. 2. Tracheal epithelium after 6 weeks of DEN-treatment: basal cells have started to proliferate. x 600.
Fig. 3. Middle layer of hyperplastic tracheal epithelium after 8 weeks of DEN-treatment: This area is occupied by cells which appear transitional in their differentiation between basal and mucous cells. x 12 000.
188 . Hildegard M. Reznik-Schuller
Fig. 4. Trachea in the 13th week of DEN-treatment: papillary polyp has developed, the basal part of which is shown in this picture. X 8000.
Fig. 5. Part of papillary polyp after 14 weeks of DEN-treatment: The cytoplasm of a mucous cell contains many mucous granules. x 6000.
Pathogenesis of Diethylnitrosamine-Induced Tumors· 189
Fig. 6. Squamous metaplasia in another part of the papillary polyp shown in Fig. 5: The cytoplasm contains thick bundles of filaments while other cell organelles are scanty. x 18300.
Fig. 7. Squamous carcinoma in the trachea after 34 weeks of DEN-treatment: the tumor cells have invaded the subepithelial tissue and cartilage. X 100.
190 . Hildegard M. Reznik-Schiiller
ing the basal layer. The middle layer of such hyperplastic areas was occupied by cells which seemed to be transitional in their differentiation between basaland mucous cells (Fig. 3). They did contain a well-developed endoplasmic reticulum, like mucous cells, but unlike the latter, did not show any production of mucous. The surface layer of the hyperplastic epithelia was composed of fully differentiated mucous cells while ciliated cells were scanty. The first tracheal tumors, papillary polyps (Fig. 4), were seen in the 13th week and were found in all of the animals sacrificed thereafter. The neoplasms consisted almost entirely of transitional and mucous cells, while basal cells were restricted to the basal layer and ciliated cells were lacking completely. The cytoplasm of some mucous cells was occupied by mucous granules (Fig. 5). Other mucous cells, as well as some of the transitional cells, exhibited various amonts of filament bundles in their cytoplasm, which indicates that these cells were undergoing squamous metaplasia (Fig. 6). Two of the four animals which survived more than 20 weeks had squamous carcinomas in addition. These neoplasms invaded the subepithelial tissue and cartilage (Fig. 7). At the ultrastructural level such tumor cells were characterised by abundant cytoplasmic filaments and sparseness of other cytoplasmic organelles. During the entire period of observation, no proliferation of APUD-type cells was seen in the tracheal epithelium.
Discussion Previous sequential studies of the development of DEN-induced pulmonary tumors in Syrian hamsters have revealed early ultrastructural changes in bronchial Clara cells and APUD-ceIls, and these two cell types were also the sources of the neoplasms (Reznik-SchUller, 1976a, b, 1977). The high content of oxidative enzymes in Clara cells (Azzopardi and Thurlbeck, 1969), together with the ability of APUD-cells to take-up and metabolize simple amines and their precursors (Pearse, 1974), lead us to the conclusion that metabolic competence is an important factor in the selective action of nitrosamines (ReznikSchUller and Lijinsky, 1979). In view of this, mucous cells would seem the most likely site for nitrosamine activation in the tracheal epithelium because they are metabolically the most active cells in this area (Azzopardi and Thurlbeck, 1968). It is surprising, therefore, that it was the metabolically most inactive epithelial cells (Kaufmann et al. 1972), the basal cells, that were the origin of neoplastic development in the trachea although these cells were not affected during the initial treatment period. When one searches for common characteristics of cell types which produce tumors in both bronchi and trachea, it becomes evident that bronchial Clara
Pathogenesis of Diethylnitrosamine-Induced Tumors· 191
cells and tracheal basal cells are both stem cells for cell renewal (Evans et aI., 1976; Shorter, Titus and Divertie, 1964). In the bronchus, one target cell of DEN is a stem cell, the Clara cell, which is metabolically very active, while in the trachea, the target cell is also a stem cell, the basal cell, which is metabolically inactive. This suggests that while metabolic competence is important, it is not the only factor. For cells to be capable of undergoing neoplastic change, a preexisting ability to proliferate seems important. It is also possible that the ultimate carcinogenic metabolite of the nitrosamine is formed in another cell type, in the immediate vicinity of the target Clara or basal cells (e.g. in mucous cell in the trachea). From our present knowledge, this is a rather theoretical consideration. We require further information on the intracellular site of binding and metabolism of the inducing carcinogen to settle this problem. Acknowledgements. The authors would like to thank Ms. Lee Ann Nutter for excellent technical assistance. This work was initiated at the section of Experimental Pathology of the Medical University, Hannover (FRG).
References Azzopardi, A., and Thurlbeck, W. M.: Oxidative enzyme pattern of the bronchial mucous glands. Amer. Rev. resp. Dis. 97, 1038-1045 (1968) Azzopardi, A., and Thurlbeck, W. M.: The histochemistry of the nonciliated bronchiolar epithelial cell. Amer. Rev. resp. Dis. 99, 516-525 (1969) Becci, P. J., McDowell, E. M., and Trump, B. F.: The respiratory epithelium. II. Hamster trachea, bronchus, and bronchioles. J. mat. Cancer Inst. 61, 551-562 (1978) Dontenwill, W., and Mohr, U.: Carcinome des Respirationstraktes nach Behandlung von Goldhamstern mit Diaethylnitrosamin. Z. Krebsforsch. 64, 305-312, (1961) Dontenwill, W., Mohr, U., and Zagel, M.: Uber die unterschiedliche Lungencarcinogene Wirkung von Diaethylnittosamin bei Hamster und Ratte. Z. Krebsforsch. 64, 499-502 (1962) Evans, M. J., Johnson, L. V., Stephens, R. J., and Freeman, G.: Renewal of the terminal bronchiolar epithelium in the rat following exposure to N0 2 or Os. Lab. Invest. 35, 246-257 (1976) Herrold, J. M.: Effect of route of administration on the carcinogenic action of diethylnitrosamine (N-nitroso-diethylamine). Brit. J. Cancer 18, 763-767 (1964) Herrold, K. M., and Dunham, L.: Induction of tumors in the Syrian Golden hamster with diethylnitrosamine (N-N-nitrosodiethylamine). Cancer Res. 23, 773-777 (1963) Kaufman, D. G., Baker, M. S., Harris, C. c., Smith, J. M., Boren, H., Sporn, M. B., and Saffiotti, U.: Co-ordinated biochemical and morphological examination of hamster tracheal epithelium. J. nat. Cancer Inst. 49, 783-792, (1972) Montesano, R., and Saffiotti, U.: Carcinogenic response of the respiratory tract of Syrian Golden hamsters to different doses of diethylnitrosamine. Cancer Res. 28, 2197-2210 (1968) Pearse, A. G. E.: The APUD cell concept and its implications in pathology. Path. Annu. 9, 27-41 (1974) Reznik-Schuller, H.: Proliferation of endocrine (APUD-type) cells during early N-diethylnitrosamine-induced lung carcinogenesis in hamsters. Cancer Lett. 1, 255-258 (1976a)
192 . Hildegard M. Reznik-SchUller Reznik-Schiiller, H.: Ultrastructural alterations of nonciliated cells after nitrosamine treatment and their significance for pulmonary carcinogenesis. Amer. J. Path. 85, 549-552 (1976b) Reznik-Schiiller, H.: Ultrastructural alterations of APUD-cells during nitrosamine-induced lung carcinogenesis. J. Path. 121, 79-82 (1977) Reznik-Schiiller, H., and Lijinsky, W.: In vivo autoradiography and nitrosoheptamethyleneimine carcinogenesis in hamsters. Cancer Res. 39, 72-74, (1979) Shorter, R. G., Titus, J. L., and Divertie, M. B.: Cytodynamics in the respiratory tract. Dis. Chest
46, 138-142 (1964)
Received July 2, 1979 . Accepted August 17, 1979
Key words: Diethylnitrosamine - Carcinogenesis - Hamster - Trachea Ultrastructure - Mucous cells
Dr. Hildegard M. Reznik-SchUller, NCI Frederick Cancer Research Center, Frederick, MD,
21701, USA