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Autophagic mechanisms in diethylnitrosamine-induced tracheal papillomas of Syrian golden hamster
Exp. Pathol. 32, 55--60 (1987) VEB Gustav Fischer Verlag J ena Institut Ifir Experimente lle Path ologie, Medizinische Hochschule Hanno ver, F.R. G.
Autophagic mechanisms in diethylnitrosamine-induced tracheal papillomas of Syrian golden hamster By U. WAHNSCHAFFE, M. EMURA and U. :MOHR With 6 figures
Address for correspondence: Prof. Dr. U. MO HR , Institut fiir Experimentelle Pathologie, Medizinische Hochschule Hann over, Konstant y-Gutschow-Str aBe 8, D - 3000 Hanno ver 61, F.R. G. K e y wo r ds : diet hylnitros amine ; tr acheal papillomas ; lysosomes ; giant lysosomes; aut ophagocyt osis; ultr astructure
Summary Tr eatment wit h dieth ylnitrosamine induced tracheal papillomas in the Syrian golden ham st er. E nlarged lysosomes (E L) occurred in a particular t yp e of cells sit uate d at the peripher y of tu mor lobul es. The differenti at ed phen ot yp e of these cells is secreto ry in nature. As t o th e significance of E L occurrence , t wo possib iliti es are considere d: 1) EL form ati on is a part of disposal mechani sms afte r th e aut op hagocytos is of und ischarged secreto ry pr odu ct s by secondary lysosomes and 2) E Ls ar e indi cative of sublet hal injury to cells of the outermost layer of t ra cheal papill omas.
Introduction Previous reports have postulated lysosomal involvement in the regulation of cellular secretio n in ante rior pituitary glands (SMITH and FARQ UHAR 1966 ; F ARQUHAR 1969). Th ey described accumulations of undischar ged secretory. gra nules, which were disposed by auto phagy int o lysosomes. In a recent st udy ( ~IA T TSON et al. 1986), cult ured tumor cells of t he same gland showed " giant" lysosomes st imulate d for auto phagocyto sis with
ACTH. Th e present paper describ es a similar auto phagi c ph enomenon in cells with secreto ry differenti ation in tracheal pap illary tum ors of the Syria n golden ham st er (SG H) induced by N-diethy l-nit rosa mine (DEN) . Since det ailed morph ological descripti on of this autophagy is lacking, we combined light and elect ron microscopy with different hist ochemi cal methods t o investigate ly sosomal for mation and digesti on and their r elationship t o cell type and st age of diff erentiation.
Materials and Methods Tra cheal neoplasm from 5 male and 8 female Syrian golden hamste rs (SGII) aged 51-54 weeks was invest igat ed. The animals were housed under standard laboratory conditions and received food and wat er ad libitum. Weekly s.c. inj ection of animal s with 20 mg/kg body weight DEN (LD5o = 178 mg/k g b.w.) was started at th e age of 24 weeks. DE N (98 % purity) was kindl y provided by Prof. PREUSSMAN, Deut sches Krebsforschun gszentrum, Heidelberg, F.R.G. Twent y-seven to 34 weeks after th e st art of injection, th e animals were anaesthetised. Three males and 5 females were perfused with 2 % cacodylate -buffered glutaraldehyd e (p H = 7.4; 330 mosm), Whole tracheas of 2 males and 3 females were fixed by immersion in Bouin's fixative. These immersion -fixed tissues with microscopically-visible tumors were dehydrated and embedded in 2-hydro xy ethylmethacrylate (Technovit 7100) and 2 p,m sections were stained with Stev enel's blue (DEL CERRO 1980), periodic-acid-Schiff (PAS)-hematoxylin or alcian blu e (p H = 2.5) (AB).
55
Tissues fixed by perfusion were osmicated, dehydrated and embedded in Epon. Semithin sections were stained with Stevenel's blue, viewed under a light microscope and reembedded according to WAHNSCHAFFE (1985). Ultrathin sections were stained with uranyl acetate and lead nitrate and viewed under a Zeiss EM 10 electron microscope.
Results Weekly treatment with DEN induced papillary tumors (100% incidence) in the trachea of all 13 hamsters. The morphology of these tumors has been described by several authors (ALTHOFF et al. 1971; REZNIK-SCHULLER 1980; MOHR 1982; RICHTER-REICHHELM et al. 1985). In 85 % of all tumors investigated light microscopically (n = 78), intracytoplasmic spherical bodies occurred (up to 10/tm in diameter) which, for the purposes of this study, we named enlarged lysosomes (EL). These ELs were found exclusively in neoplastic cells of the outermost layer of tumor lobes and appeared dark blue in semithin Epon or Technovit sections stained with Stevenel's blue. In parallel semithin Technovit sections, the ELs showed no positive reaction to the staining with PAS alone, PAS-AB or AB alone, while mucus granules were stained red or deep red and intracytoplasmic lumens blue or purple (WAHNSCHAFFE et al., submitted). At higher magnifications of semithin Epon sections, light blue, round inclusions (1~2/lm in diameter) were visible in the ELs (fig. 2). Neoplastic cells containing ELs occurred with different features: (1) the cytoplasm contained PAS-positive granules; (2) besides ELs, intracytoplasmic lumens filled with secreted compounds (figs 1, 8) were present or (3) the cytoplasm showed no positive reaction for mucus substances. As to case (1), the small round inclusions in the ELs were also PASpositive. Electron microscopically, cells with ELs showed multilobulated nuclei with heterochromatins localized at the nuclear periphery. Mitochondria occurred frequently, and Golgi apparatuses and endoplasmic reticulum were well developed (fig. 4), suggesting production of secretory materials. Some ofthese cellsshowedsecretory vacuoles (O.8~ 1.5 /lm in diameter, fig. 6) filled with mucus substances as evident from the PAS staining. Variously-developing lysosomes were found at different digestive stages. They arose originally through fusion of Golgi vesicles (primary lysosomes) and contained a fine, granular, electron dense matrix (fig. 3). Primary and secondary lysosomes apparently coalesced together and ingested target vesicles, giving rise to ELs sized up to lO/tm in diameter. ELs incorporated Golgi
Figs 1 and 2. Light micrographs of Epon semithin sections stained with Stevenel's blue. Fig. 1 shows cell bearing lysosomes (arrow) in the outermost layer of tumor lobules. Tracheal lumen (T), intracytoplasmic lumen (arrowhead), mucus granules (double arrowhead). x 800. Fig. 2 shows light inclusions (arrows) visible in the lysosomes. Tracheal lumen (T). x 1,070.
56
Exp. Pathol. 32 (1987) 1
Figs 3-8. Electron micrographs of ultr athin sections stained wit h uran yl acetat e and lead cit rate, Fig. 3 shows secretory vac uoles (double arrowhead) engulfed by lysosomes (ly). Golgi apparatus (G), Golgi vesicles (arrowhead), nu cleus (n), ext racellular space (E) . x 18,000. Fig. 4 shows lysosome (ly) in fusion wit h Golgi vesicles. Golgi apparatus (G), secret ory vacuoles (v). X 36,000. Fig. 5. Lysosomes (ly) incorporatin g a droplet of cytosol (arrow). X 18,300. Fig. 6. Lysosomes pa cked with pleomorph ic mat erials (arr ow). Nucleus (n), secretory va cuoles (arrowhead). x 4,800.
Fig. 7. Terminal stage of digestion. Electron dense remnants (arrows) in electron transparent space. Lysosome (ly), intracytoplasmic lumen (ic). x 8,100. Fig. 8. Golgi vesicles (arrow) being incorporated by lysosome (ly). Golgi apparatus (G).
58
Exp. Pathol. 32 (1987) 1
x 61,000.
vesicles with unknown contents (fig. 7), secretory vacuoles (fig. 4) and cytosol without limiting membranes (fig. 5). Complex organelles, such as mitochondria, appeared to be seldom engulfed in the ELs. The active phase of digestion in ELs is indicated by increased electron density (membraneous fragments) and electron lucent spherical spaces. Some ELs were packed with digestive, pleomorphic materials (fig. 6). At the terminal stage of digestion, a few electron dense remnants were seen in electron transparent spaces left amid the cytoplasm (fig. 8), often being devoid of limiting membranes. None of the cells bearing ELs showed any sign of degeneration.
Discussion The ELs described in the present study were apparently derived from the autophagic secondary lysosomes. They occurred to a much larger extent in a secretory type of cell constituting the tracheal papillary tumors and to a much smaller extent in cells of normallooking tracheal epithelia of untreated animals. This phenomenon has not been described in previous investigations (ALTHOFF et al. 1971; REZNIK-SCHULLER 1980; RICHTER-REICHHELM et al. 1985) on tracheal papillary tumors of SGHs. This is probably because we investigated by contrast the EM sections obtained directly from light microscopically controlled and documented semithin sections (WAHNSCHAFFE 1985) in combination with various histochemical methods carried out on semithin Technovit sections. Secondary lysosomes of similar sizes were described in macrophages phagocytosing cellular debris of degenerating neurons (CHU-WANG and OPPENHEIM 1978) or in those involved in morphogenetic cell death (WEBER 1969; WAHNSCHAFFE et al. in press). In our case, we observed neither phagocytosis nor advanced endocytosis by our tumor secretory cells (usually indicated by the presence of clathrin-coated vesicles), so we presume that an autophagocytosis occurred, mostly of secretory vesicles produced by the Golgi apparatuses. Similar authophagic processes have been described in the disposal of undischarged secretory granules in cells of the anterior pituitary gland (FARQUHAR 1969) and in endocrine A-cells of the pancreas of animals with experimental diabetes (ORC! et al. 1968). MATTSON and coworkers (1986) observed "giant" lysosomes in cultured adrenal tumor cells after stimulation with ACTH. As to the significance of occurrence of ELs, several possibilities may be considered. For as yet unknown reasons, the secretory vesicles produced by the ER and Golgi apparatuses were not secreted outside the tumor cells, 80 that the autophagic mechanism appeared to take over the disposal of accumulating secretory products leading to the formation of ELs. Another possible mechanism occurring simultaneously (fig. 1) would be the development of intracytoplasmic lumens (WAHNSCHAFFE et al., submitted.) in which the therein secreted materials could be stored and eventually decomposed. Yet another possibility could be that the cells situated at the periphery of tumor lobules were sublethally injured (Dr. KEENAN, personal communication) due to some reasons such as anoxia, malnutrition and/or even toxicity of weekly-administered DEN and their organelles were thus undergoing internal turnover, as was also the ease in the hepatic parenchymal cells injured sublethally by a toxicant (KERR 1973). In particular, the occurrence of ELs in cells possessing well-developed ER and Golgi apparatuses but no PAS-positive material at the tumor periphery can be explained by their enhanced carcinogen-metabolising capability (MOHR et al. 1979; EMURA et al. in press) which renders them vulnerable to the toxic metabolites of DEN. In any case, the formation of ELs represents a pathological situation partly related to a distorted function of neoplastic cells (MATTSON et al. 1986) and partly due to sublethal injury to cells.
References J., WILSON, R., MOHR, D.: Diethylnitrosamine-induced alterations in the tracheobronchial system of Syrian golden hamster. J. Nat!. Cancer Inst, 1971; 46: 1067-1071.
ALTHOFF,
Exp. Pathol. 32 (1987) 1
59
CHU-WANG, 1.-W., OPPENHEIM, R.: Cell death of motoneurons in the chick embryo spinal cord. J. Compo Neur. 1978; 177: 33-58. DELCERRO, M., COGEN, J., DEL CERRO, C.: Stevenel's blue, an excellent stain for optical study of plastic embedded tissues. Microscopia Acta 1980; 83: 117-121. EMURA, M., MOHR, D., RIEBE, M., AUFDERHEIDE, M., DUNGWORTH, D. L.: Predisposition of cloned fetal hamster lung epithelial cells to transformation by a pre carcinogen, benzo(a)pyrene, using growth hormone supplementation and collagen gel substratum. Cancer Res., in press. FARQUHAR, M. G.: Lysosome function in regulating secretion: disposal of secretory granules in cells of the anterior pituitary gland. In: Lysosomes in Biology and Pathology 2 (eds. J. DINGLE and H. FELL). North-Holland Publishing Company, 1969; pp 462-482. KERR, J. F.: Some lysosome function in liver cells reaching to sublethal injury. In: Lysosomes in Biology and Pathology 3 (ed. J. DINGLE). North- Holland Publishing Company. 1973; pp. 365394. MATTSON, P., LORENZ, S., KOWAL, J.: The effect of ACTH on acid phosphatase activity in endosomes, GERL and lysosomes of cultured adrenal tumor cells. Virchows Archiv B 1986; 1)1: 137-153. MOHR, D.: Tumors of the respiratory tract. In: Pathology of Tumors in Laboratory Animals (ed. V. TURUSOV). IARC Scientific Publications No 34, Lyon 1982; pp. 115-146. - REZNIK-SCHULLER, H., EMURA, M.: Tissue differentiation as a prerequisite for transplacental carcinogenesis in the hamster respiratory system, with specific respect to the trachea. Natl. Cancer Inst. Monogr. 1979; 1)1: 117-122. ORCI, L., JUNOD, A., PICTET, R., RENOLD, A., ROUILLER, C.: Granulolysis in A cells of endocrine pancreas in spontaneous and experimental diabetes in animals. J. Cell Biol, 1968; 38: 462-466. REZNIK-SCHULLER, H.: Pathogenesis of diethylnitrosamine-induced tumors in the trachea of the Syrian golden hamster. Path. Res. Pract. 1980; 168: 185-192. RICHTER-REICHHELM, H.-B., BONING, W., und ALTHOFF, J.: Response to carcinogens of respiratory epithelium, Syrian golden hamster (Mesocricetus auratus). In: Respiratory System (eds. T.C. JONES et al.). Springer Verlag, New York 1985; pp 33-40. SMITH, R. E., FARQUHAR, M. G.: Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland. J. Cell Biol, 1966; 31: 319-347. WAHNSCHAFFE, D.: A method for reembedding semithin sections for ultrahin sectioning. Mikroskopie 1985; 42: 108-110. - BARTSCH, D., FRITZSCH, B.: Metamorphic changes within the lateral-line system of anura. J. Anatomy and Embryology, in press. - EMURA, M., MOHR, D.: Development of intracytoplasmic lumen in diethylnitrosamine-induced tracheal papillomas of Syrian golden hamster. A light and electron microscopical study. Virchows Archiv B, submitted. WEBER, R.: Tissue involution and lysosomal enzymes during anuran metamorphosis. In: Lysosomes in Biology and Pathology 2 (cds. J. DINGLE and H. FELL). North-Holland Publishing Company 1969: pp. 437-461. (Received January 22, 1987)
60
Exp. Pathol. 32 (1987) 1
Autophagic mechanisms in diethylnitrosamine-induced tracheal papillomas of Syrian golden hamster By U. WAHNSCHAFFE, M. EMURA and U. :MOHR With 6 figures
Address for correspondence: Prof. Dr. U. MO HR , Institut fiir Experimentelle Pathologie, Medizinische Hochschule Hann over, Konstant y-Gutschow-Str aBe 8, D - 3000 Hanno ver 61, F.R. G. K e y wo r ds : diet hylnitros amine ; tr acheal papillomas ; lysosomes ; giant lysosomes; aut ophagocyt osis; ultr astructure
Summary Tr eatment wit h dieth ylnitrosamine induced tracheal papillomas in the Syrian golden ham st er. E nlarged lysosomes (E L) occurred in a particular t yp e of cells sit uate d at the peripher y of tu mor lobul es. The differenti at ed phen ot yp e of these cells is secreto ry in nature. As t o th e significance of E L occurrence , t wo possib iliti es are considere d: 1) EL form ati on is a part of disposal mechani sms afte r th e aut op hagocytos is of und ischarged secreto ry pr odu ct s by secondary lysosomes and 2) E Ls ar e indi cative of sublet hal injury to cells of the outermost layer of t ra cheal papill omas.
Introduction Previous reports have postulated lysosomal involvement in the regulation of cellular secretio n in ante rior pituitary glands (SMITH and FARQ UHAR 1966 ; F ARQUHAR 1969). Th ey described accumulations of undischar ged secretory. gra nules, which were disposed by auto phagy int o lysosomes. In a recent st udy ( ~IA T TSON et al. 1986), cult ured tumor cells of t he same gland showed " giant" lysosomes st imulate d for auto phagocyto sis with
ACTH. Th e present paper describ es a similar auto phagi c ph enomenon in cells with secreto ry differenti ation in tracheal pap illary tum ors of the Syria n golden ham st er (SG H) induced by N-diethy l-nit rosa mine (DEN) . Since det ailed morph ological descripti on of this autophagy is lacking, we combined light and elect ron microscopy with different hist ochemi cal methods t o investigate ly sosomal for mation and digesti on and their r elationship t o cell type and st age of diff erentiation.
Materials and Methods Tra cheal neoplasm from 5 male and 8 female Syrian golden hamste rs (SGII) aged 51-54 weeks was invest igat ed. The animals were housed under standard laboratory conditions and received food and wat er ad libitum. Weekly s.c. inj ection of animal s with 20 mg/kg body weight DEN (LD5o = 178 mg/k g b.w.) was started at th e age of 24 weeks. DE N (98 % purity) was kindl y provided by Prof. PREUSSMAN, Deut sches Krebsforschun gszentrum, Heidelberg, F.R.G. Twent y-seven to 34 weeks after th e st art of injection, th e animals were anaesthetised. Three males and 5 females were perfused with 2 % cacodylate -buffered glutaraldehyd e (p H = 7.4; 330 mosm), Whole tracheas of 2 males and 3 females were fixed by immersion in Bouin's fixative. These immersion -fixed tissues with microscopically-visible tumors were dehydrated and embedded in 2-hydro xy ethylmethacrylate (Technovit 7100) and 2 p,m sections were stained with Stev enel's blue (DEL CERRO 1980), periodic-acid-Schiff (PAS)-hematoxylin or alcian blu e (p H = 2.5) (AB).
55
Tissues fixed by perfusion were osmicated, dehydrated and embedded in Epon. Semithin sections were stained with Stevenel's blue, viewed under a light microscope and reembedded according to WAHNSCHAFFE (1985). Ultrathin sections were stained with uranyl acetate and lead nitrate and viewed under a Zeiss EM 10 electron microscope.
Results Weekly treatment with DEN induced papillary tumors (100% incidence) in the trachea of all 13 hamsters. The morphology of these tumors has been described by several authors (ALTHOFF et al. 1971; REZNIK-SCHULLER 1980; MOHR 1982; RICHTER-REICHHELM et al. 1985). In 85 % of all tumors investigated light microscopically (n = 78), intracytoplasmic spherical bodies occurred (up to 10/tm in diameter) which, for the purposes of this study, we named enlarged lysosomes (EL). These ELs were found exclusively in neoplastic cells of the outermost layer of tumor lobes and appeared dark blue in semithin Epon or Technovit sections stained with Stevenel's blue. In parallel semithin Technovit sections, the ELs showed no positive reaction to the staining with PAS alone, PAS-AB or AB alone, while mucus granules were stained red or deep red and intracytoplasmic lumens blue or purple (WAHNSCHAFFE et al., submitted). At higher magnifications of semithin Epon sections, light blue, round inclusions (1~2/lm in diameter) were visible in the ELs (fig. 2). Neoplastic cells containing ELs occurred with different features: (1) the cytoplasm contained PAS-positive granules; (2) besides ELs, intracytoplasmic lumens filled with secreted compounds (figs 1, 8) were present or (3) the cytoplasm showed no positive reaction for mucus substances. As to case (1), the small round inclusions in the ELs were also PASpositive. Electron microscopically, cells with ELs showed multilobulated nuclei with heterochromatins localized at the nuclear periphery. Mitochondria occurred frequently, and Golgi apparatuses and endoplasmic reticulum were well developed (fig. 4), suggesting production of secretory materials. Some ofthese cellsshowedsecretory vacuoles (O.8~ 1.5 /lm in diameter, fig. 6) filled with mucus substances as evident from the PAS staining. Variously-developing lysosomes were found at different digestive stages. They arose originally through fusion of Golgi vesicles (primary lysosomes) and contained a fine, granular, electron dense matrix (fig. 3). Primary and secondary lysosomes apparently coalesced together and ingested target vesicles, giving rise to ELs sized up to lO/tm in diameter. ELs incorporated Golgi
Figs 1 and 2. Light micrographs of Epon semithin sections stained with Stevenel's blue. Fig. 1 shows cell bearing lysosomes (arrow) in the outermost layer of tumor lobules. Tracheal lumen (T), intracytoplasmic lumen (arrowhead), mucus granules (double arrowhead). x 800. Fig. 2 shows light inclusions (arrows) visible in the lysosomes. Tracheal lumen (T). x 1,070.
56
Exp. Pathol. 32 (1987) 1
Figs 3-8. Electron micrographs of ultr athin sections stained wit h uran yl acetat e and lead cit rate, Fig. 3 shows secretory vac uoles (double arrowhead) engulfed by lysosomes (ly). Golgi apparatus (G), Golgi vesicles (arrowhead), nu cleus (n), ext racellular space (E) . x 18,000. Fig. 4 shows lysosome (ly) in fusion wit h Golgi vesicles. Golgi apparatus (G), secret ory vacuoles (v). X 36,000. Fig. 5. Lysosomes (ly) incorporatin g a droplet of cytosol (arrow). X 18,300. Fig. 6. Lysosomes pa cked with pleomorph ic mat erials (arr ow). Nucleus (n), secretory va cuoles (arrowhead). x 4,800.
Fig. 7. Terminal stage of digestion. Electron dense remnants (arrows) in electron transparent space. Lysosome (ly), intracytoplasmic lumen (ic). x 8,100. Fig. 8. Golgi vesicles (arrow) being incorporated by lysosome (ly). Golgi apparatus (G).
58
Exp. Pathol. 32 (1987) 1
x 61,000.
vesicles with unknown contents (fig. 7), secretory vacuoles (fig. 4) and cytosol without limiting membranes (fig. 5). Complex organelles, such as mitochondria, appeared to be seldom engulfed in the ELs. The active phase of digestion in ELs is indicated by increased electron density (membraneous fragments) and electron lucent spherical spaces. Some ELs were packed with digestive, pleomorphic materials (fig. 6). At the terminal stage of digestion, a few electron dense remnants were seen in electron transparent spaces left amid the cytoplasm (fig. 8), often being devoid of limiting membranes. None of the cells bearing ELs showed any sign of degeneration.
Discussion The ELs described in the present study were apparently derived from the autophagic secondary lysosomes. They occurred to a much larger extent in a secretory type of cell constituting the tracheal papillary tumors and to a much smaller extent in cells of normallooking tracheal epithelia of untreated animals. This phenomenon has not been described in previous investigations (ALTHOFF et al. 1971; REZNIK-SCHULLER 1980; RICHTER-REICHHELM et al. 1985) on tracheal papillary tumors of SGHs. This is probably because we investigated by contrast the EM sections obtained directly from light microscopically controlled and documented semithin sections (WAHNSCHAFFE 1985) in combination with various histochemical methods carried out on semithin Technovit sections. Secondary lysosomes of similar sizes were described in macrophages phagocytosing cellular debris of degenerating neurons (CHU-WANG and OPPENHEIM 1978) or in those involved in morphogenetic cell death (WEBER 1969; WAHNSCHAFFE et al. in press). In our case, we observed neither phagocytosis nor advanced endocytosis by our tumor secretory cells (usually indicated by the presence of clathrin-coated vesicles), so we presume that an autophagocytosis occurred, mostly of secretory vesicles produced by the Golgi apparatuses. Similar authophagic processes have been described in the disposal of undischarged secretory granules in cells of the anterior pituitary gland (FARQUHAR 1969) and in endocrine A-cells of the pancreas of animals with experimental diabetes (ORC! et al. 1968). MATTSON and coworkers (1986) observed "giant" lysosomes in cultured adrenal tumor cells after stimulation with ACTH. As to the significance of occurrence of ELs, several possibilities may be considered. For as yet unknown reasons, the secretory vesicles produced by the ER and Golgi apparatuses were not secreted outside the tumor cells, 80 that the autophagic mechanism appeared to take over the disposal of accumulating secretory products leading to the formation of ELs. Another possible mechanism occurring simultaneously (fig. 1) would be the development of intracytoplasmic lumens (WAHNSCHAFFE et al., submitted.) in which the therein secreted materials could be stored and eventually decomposed. Yet another possibility could be that the cells situated at the periphery of tumor lobules were sublethally injured (Dr. KEENAN, personal communication) due to some reasons such as anoxia, malnutrition and/or even toxicity of weekly-administered DEN and their organelles were thus undergoing internal turnover, as was also the ease in the hepatic parenchymal cells injured sublethally by a toxicant (KERR 1973). In particular, the occurrence of ELs in cells possessing well-developed ER and Golgi apparatuses but no PAS-positive material at the tumor periphery can be explained by their enhanced carcinogen-metabolising capability (MOHR et al. 1979; EMURA et al. in press) which renders them vulnerable to the toxic metabolites of DEN. In any case, the formation of ELs represents a pathological situation partly related to a distorted function of neoplastic cells (MATTSON et al. 1986) and partly due to sublethal injury to cells.
References J., WILSON, R., MOHR, D.: Diethylnitrosamine-induced alterations in the tracheobronchial system of Syrian golden hamster. J. Nat!. Cancer Inst, 1971; 46: 1067-1071.
ALTHOFF,
Exp. Pathol. 32 (1987) 1
59
CHU-WANG, 1.-W., OPPENHEIM, R.: Cell death of motoneurons in the chick embryo spinal cord. J. Compo Neur. 1978; 177: 33-58. DELCERRO, M., COGEN, J., DEL CERRO, C.: Stevenel's blue, an excellent stain for optical study of plastic embedded tissues. Microscopia Acta 1980; 83: 117-121. EMURA, M., MOHR, D., RIEBE, M., AUFDERHEIDE, M., DUNGWORTH, D. L.: Predisposition of cloned fetal hamster lung epithelial cells to transformation by a pre carcinogen, benzo(a)pyrene, using growth hormone supplementation and collagen gel substratum. Cancer Res., in press. FARQUHAR, M. G.: Lysosome function in regulating secretion: disposal of secretory granules in cells of the anterior pituitary gland. In: Lysosomes in Biology and Pathology 2 (eds. J. DINGLE and H. FELL). North-Holland Publishing Company, 1969; pp 462-482. KERR, J. F.: Some lysosome function in liver cells reaching to sublethal injury. In: Lysosomes in Biology and Pathology 3 (ed. J. DINGLE). North- Holland Publishing Company. 1973; pp. 365394. MATTSON, P., LORENZ, S., KOWAL, J.: The effect of ACTH on acid phosphatase activity in endosomes, GERL and lysosomes of cultured adrenal tumor cells. Virchows Archiv B 1986; 1)1: 137-153. MOHR, D.: Tumors of the respiratory tract. In: Pathology of Tumors in Laboratory Animals (ed. V. TURUSOV). IARC Scientific Publications No 34, Lyon 1982; pp. 115-146. - REZNIK-SCHULLER, H., EMURA, M.: Tissue differentiation as a prerequisite for transplacental carcinogenesis in the hamster respiratory system, with specific respect to the trachea. Natl. Cancer Inst. Monogr. 1979; 1)1: 117-122. ORCI, L., JUNOD, A., PICTET, R., RENOLD, A., ROUILLER, C.: Granulolysis in A cells of endocrine pancreas in spontaneous and experimental diabetes in animals. J. Cell Biol, 1968; 38: 462-466. REZNIK-SCHULLER, H.: Pathogenesis of diethylnitrosamine-induced tumors in the trachea of the Syrian golden hamster. Path. Res. Pract. 1980; 168: 185-192. RICHTER-REICHHELM, H.-B., BONING, W., und ALTHOFF, J.: Response to carcinogens of respiratory epithelium, Syrian golden hamster (Mesocricetus auratus). In: Respiratory System (eds. T.C. JONES et al.). Springer Verlag, New York 1985; pp 33-40. SMITH, R. E., FARQUHAR, M. G.: Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland. J. Cell Biol, 1966; 31: 319-347. WAHNSCHAFFE, D.: A method for reembedding semithin sections for ultrahin sectioning. Mikroskopie 1985; 42: 108-110. - BARTSCH, D., FRITZSCH, B.: Metamorphic changes within the lateral-line system of anura. J. Anatomy and Embryology, in press. - EMURA, M., MOHR, D.: Development of intracytoplasmic lumen in diethylnitrosamine-induced tracheal papillomas of Syrian golden hamster. A light and electron microscopical study. Virchows Archiv B, submitted. WEBER, R.: Tissue involution and lysosomal enzymes during anuran metamorphosis. In: Lysosomes in Biology and Pathology 2 (cds. J. DINGLE and H. FELL). North-Holland Publishing Company 1969: pp. 437-461. (Received January 22, 1987)
60
Exp. Pathol. 32 (1987) 1