Ultrastructural Alterations Produced by Polycyclic Aromatic Hydrocarbons on Rat Trachã©Alepithelium in Organ Culture1

[CANCER RESEARCH 28, 90&-923, May 1968]

Ultrastructural Alterations Produced by Polycyclic Aromatic Hydrocarbons on Rat TrachÃ©alEpithelium in Organ Culture1

E. Roter Dirksen and T. Timothy Crocker Cancer Research Institute and Department o] Medicine, School oj Medicine, University oj California San Francisco Medical Center, San Francisco, California 94122

SUMMARY permit penetration and increase the probability of biologic interaction. Moreover, only local responses of tissue to test Ultrastructural alterations of columnar respiratory epithe compounds occur, thus providing an evaluation of direct local lium were produced by certain polycyclic aromatic hydrocar effects rather than of indirect effects arising from metabolites bons in suckling rat trachea maintained in organ culture for produced in the whole animal. The response of target cells can 11 days. The potent carcinogens 7,12-dimethylbenz(a)anthra- be visualized without secondary inflammatory or other re cene (DMBA) and benzo(a)pyrene and the weakly carci sponses which would arise in the intact animal. nogenic compounds benz(a)anthracene and 5-fluoro-7,12-di- In previous histologie and autoradiographic studies of the methylbenz(a)anthracene (F-DMBA) produced cells whose effects of polycyclic aromatic hydrocarbons on suckling rat cytoplasm contained little endoplasmic reticulum, many free trachea in organ culture, methylcholanthrene, benzo(a)py- ribosomes, complex autophagic vacuoles, and abundant cyto- rene, benz(a)anthracene, and 7,12-dimethylbenz(a)anthracene plasmic filaments. These cells were often connected by many (DMBA) produced abnormal states of epithelial differentia desmosomes and complex interdigitations in a stratified epi tion and increased the proportions of basal cells labeled with thelium 3 to 8 cells high, which bore a similarity to stratified tritiated thymidine (14, 15). These hydrocarbons are estab squamous epithelia such as skin and also resembled broncho- lished carcinogens of varying potency in rodents (22, 29, 43) genie carcinoma in situ. Some nucleolar fragmentation and dis and for the sake of brevity will be referred to as carcinogenic organization also occurred in the epithelium of the carcinogen- hydrocarbons. treated expiants. In general, DMBA produced morphologic One additional compound has been studied in organ culture abnormalities at low concentrations, while the related com by histologie and autoradiographic methods and requires spe pounds benz(a)anthracene and F-DMBA did so at concentra cial consideration. The fluoro analog of DMBA, 5-fluoro-7,12- tions 12.5- to 25-fold higher. Although autophagic vacuoles dimethylbenz(a)anthracene (F-DMBA) has been reported as and related structures, as well as some degree of disorganization "not carcinogenic (or very weakly so)" by Bergmann et al. of the endoplasmic reticulum, were found in columnar cells, (7). J. A. Miller and E. C. Miller compared the carcinogenic and increased filaments were noted in basal cells, extensive al activity of DMBA and F-DMBA in three systems (unpub teration of cell structure and epithelial state was not encoun lished observations). They report that, when adult female rats tered in control expiants or in expiants treated with anthracene, were injected subcutaneously with 2.12 mg DMBA or 2.26 mg benzo(e)pyrene, benzo(a)fluorene, benz(m,n,o)fluoranthene, F-DMBA in trioctanoin, all 18 DMBA-treated and 3 of 16 chrysene, perinaphthoxanthene, perylene, or phenanthrene. F-DMBA-treated rats developed sarcomas by 14 months, while the trioctanoin controls did not. When the skin of adult female INTRODUCTION mice was painted (twice weekly for 40 weeks) with 70 /Â¿g DMBA or 79 fig F-DMBA in acetone, by 15 months carcinoma Many ultrastructural studies have analyzed the changes of the skin had developed in 21 of 31 DMBA-treated, in 1 of produced in particular tissues by exposure of the whole an 29 F-DMBA-treated, and in none of the acetone control mice. imal to the carcinogenic polycyclic hydrocarbons or other If single applications of 159 fig or 340 /Â¿gof DMBA and F- chemical carcinogens (5, 8, 17, 20, 35, 40, 46, 57). In the present DMBA, respectively, were painted on the skin of mice, followed work, an organ culture system was used to study the early by repeated applications of croton oil (twice weekly for 36 ultrastructural changes produced by polycyclic hydrocarbons weeks), DMBA produced 138 skin papillomas in 26 of 40 mice in respiratory epithelium. There are many advantages to such and F-DMBA produced 15 papillomas in 7 of 40 mice, while a system, for tissues can be exposed to hydrocarbons of poor 2 of 40 croton oil controls developed 3 papillomas. At 48 weeks, solubility or low biologic activity for periods long enough to carcinomas had developed in 9 of the DMBA-treated and in 1 of the F-DMBA-treated mice. 1 Supported in part by Research Contract PH 43-64-42, National These data agree with our observations that F-DMBA has Cancer Institute, USPHS, and by cancer research funds of the low but demonstrable carcinogenic activity. In this laboratory, University of California. both F-DMBA and benz (a) anthracene required the same high Received August 14, 1967; accepted January 12, 1968. concentrations to produce biologic effects in rat trachÃ©alepi-

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1968 American Association for Cancer Research. Alterations of Rat TrachÃ©alEpithelium thelium in organ culture. These effects were qualitatively similar of penicillin and 5% chicken serum. A clotted medium (1 ml) to those produced by DMBA, but F-DMBA and benz (a) an was prepared as described previously (14, 16) but was allowed thracene were active only at 12.5- to 25-fold higher concentra to form in the center well of a plastic (Falcon) organ culture tions than DMBA (14-16). On the strength of the in vivo indi dish rather than in glass containers. Each dish had two strips cations for weak but definite carcinogenicity of F-DMBA and of rayon mesh. Three to five expiants, consisting of the center for benz (a) anthracene (29), and also on the basis of com third of a trachea, were placed on each strip of mesh. The parable findings for these compounds in our prior experiments, mesh with the attached expiants was placed in fresh medium F-DMBA is grouped with the carcinogenic hydrocarbons in every third day. The dishes were enclosed in an airtight box this report. fitted for intermittent gassing with a mixture of carbon dioxide Eight additional hydrocarbons were used to estimate the (3%) and oxygen (97%) (100 ml per minute for 30 minutes specificity of response of respiratory epithelium to the car daily). cinogenic hydrocarbons. These were benzo(e)pyrene, phenan- After 11 days in culture, expiants were fixed in 1.33% Os04 threne, chrysene, benzo(a)fluorene, benz(m,n,o)fluoranthene, buffered with s-collidine at pH 7.4 (6) to which 0.02% CaCl2 perylene, perinaphthoxanthene, and anthracene (14, 15). These had been added. Following an initial 15-minute period of fixa will be referred to as noncarcinogenic hydrocarbons (22, 43), tion, the expiants were placed in 1% melted agar at about although chrysene is acknowledged to be a weak carcinogen. 40Â°C. Infiltration with warm agar was desirable to prevent The state of differentiation observed in epithelium of rat tra occasional swelling or ballooning of the epithelial cells at the chÃ©alorgan cultures exposed to this group of compounds was luminal surface. After agar infiltration, the agar-tissue block not different from control cultures when appropriate systems was chilled, and the tissue was cut out of the set agar and of exposure were employed (14, 15). returned to fresh fixative for an additional hour. The tissue In the present report, fine structural changes are described was then dehydrated in a graded series of acetones, stained in from experiments performed in parallel with the studies men block with KMnO4 (38), and embedded in Araldite (31). tioned above. Histologie observations on the responsiveness of Sections were cut with a diamond knife on an MT-2 Porter- trachÃ©alepithelium to the carcinogenic hydrocarbons as com Blum ultramicrotome and were picked up on parlodion-coated, pared to the noncarcinogenic hydrocarbons were, in general, carbon-evaporated grids. They were stained with 2% uranyl confirmed by electron microscopy, but the higher level of res acetate for one hour and with lead citrate for 20 minutes (44). olution of the electron microscope permitted more detailed The sections were viewed and photomicrographs were taken analysis of early effects of biologically active polycyclic aro with an RCA-EMU 3G electron microscope, using 100 kv matic hydrocarbons, and facilitated qualitative distinctions accelerating voltage and 35-/Â¿objective apertures. between effects of the culturing technic itself and those altera tions which accumulated as an added response to the hydro OBSERVATIONS carbons. Cellular Organization and Intercellular Relationships MATERIALS AND METHODS Intact Animal. In the rat, the trachÃ©alepitheliumis simple columnar; it consists of 30% ciliated, 30% goblet, 10% The following hydrocarbons were kindly supplied by Dr. "brush", and 30% basal cells (45). The columnar cells extend Hans L. Falk of the National Cancer Institute: anthracene, from the basement membrane to the luminal surface where benz (a) anthracene, benzo(e)pyrene, phenanthrene, chrysene, the cells are connected by junctional complexes (23), and perylene, benzo(a)fluorene, benz(m,n,o)fluoranthene, and peri have nearly vertical boundaries with very little intercellular naphthoxanthene. F-DMBA, prepared by Dr. Melvin Newman space. At the basement membrane, each cell tapers to a small of Ohio State University, was supplied by Dr. James A. Miller, "foot". The spaces between the feet of columnar cells are filled University of Wisconsin. DMBA and benzo(a)pyrene were by basal cells. obtained from Mann Research Laboratories, Inc. The ultrastructural morphology of the four types of cells Each hydrocarbon was dissolved in acetone and added to of the rat trachea has been described in detail by Rhodin and chicken serum to form a stock suspension of such concentra Dalhamn (45) and is not included here. The following observa tion that one part of the stock suspension per 30 parts of tions primarily describe the maintenance of this structural complete culture medium yielded less than 0.2% acetone and organization after organ culture and the fine structural changes the following final concentrations of hydrocarbons: benz (a) produced in the presence of the various hydrocarbons. anthracene and F-DMBA, 5, 25, and 50 fig/ml; DMBA, 2.5 Control Expiants. After 11 days in culture, structural fea and 5.0 /Â¿g/ml; benzo(a)pyrene, 6 /Â¿g/ml; benzo(e)pyrene, tures of expiants did not deviate appreciably from normal 30 /ig/ml; phenanthrene, 50 /xg/ml; chrysene, 6.7 /ig/ml; and morphology. The most significant alterations in controls, pre benzo(a)fluorene, benz(m,n,o)fluoranthene, perylene, and peri sumably produced by organ culture conditions, consisted of an naphthoxanthene, 20 /ig/ml. increase in the number and pleomorphism of autophagic vacu Control media contained serum to which was added an oles, occasional dilation of endoplasmic reticulum cisternae, amount of acetone equivalent to that introduced with the a slight increase in the number of basal cells, and an increase hydrocarbons. in height of columnar cells (Fig. 1). Near the lumen there were Tracheas were removed from 1- or 2-day-old suckling Nelson junctional complexes, and below this point there were few rats and transferred to Tyrode's solution containing 100 units intercellular spaces greater than about 150A. In goblet cells,

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Golgi cisternae were well developed and maintained during were consistently found. Expiants cultured with these carci culture (Fig. 2), as was an extensive endoplasmic reticulum nogens lost most of the normal architectural features of the (Fig. 3). Ciliated cells, characteristically, had large numbers epithelium. Cuboidal cells, several layers of flattened cells (Fig. of mitochondria and little endoplasmic reticulum. Evidence 16), or stratified round cells (Fig. 21) were observed. In all for the normal physiologic state of these expiants could be in these cases large intercellular spaces became apparent, and ex ferred from the presence of differentiating cells, some of which tremely complex cellular relationships were produced (Figs. were in the process of centriolo formation (19). 16 and 27 are good examples). Cytoplasmic filaments were not found in cells near the lumen At biologically effective concentrations of F-DMBA, DMBA, of control explants, but basal cells contained some filaments or benzo(a)pyrene, a wide range of modulations in morphologic near the basement membrane. Intercellular spaces were nor character occurred. Features common to basal cells (intercel mally present around basal cells, but when spaces were bridged lular spaces, intercellular digitations, desmosomes, cytoplasmic by cell-to-cell contact, desmosomal junctions were present. filaments, little endoplasmic reticulum, and many free ribosomes) were present in cells lying on the basement membrane, Hydrocarbon-treated Expiants in a mid-zone of the epithelium, or at the luminal edge. Cells at the lumen had all the features of basal cells described above Cell Organization and Intercellular Relationships. The majority of the noncarcinogenic hydrocarbons produced very and were clearly not columnar, but they did have tight junc minor alterations in the general architecture of the cultured tions and microvilli as do differentiated columnar cells (Fig. rat trachÃ©alepithelium (Fig. 4). Many of the expiants retained 26). This combination of characteristics suggests that this layer of cells was still respiratory epithelium but with cell differentia the columnar appearance of their cells which extended, as usual, from the basement membrane to the lumen. Close to the cell tion having phenotypic features closer to basal cells than to columnar cells. However, these cells had more cytoplasmic surface, the cells were tightly connected with little intercellular filaments, desmosomes, and deep interdigitations than normal space (Figs. 4, 6), while at the basement membrane the cells basal cells and must be regarded as distinctively altered (Figs. tapered slightly. Evidence of progressive normal differentiation 8, 9, 11, 16, 21). was frequent, as can be seen in Fig. 7 in which a ciliated cell of a chrysene-treated expiant is forming centrioles (19). Cul Evidence of local cytoplasmic degenerative changes were en tures were occasionally obtained in which many of the cyto- countered occasionally in cells of control cultures and of cul tures treated with the noncarcinogenic hydrocarbons. Single plasmic components had been lost, most likely as the result cells of this type might be found in any culture among cells of variable culture conditions. As an example of this effect, of normal morphology. Focal cytoplasmic degeneration occurs benz(m,n,o)fluoranthene produced variable results; two types at an early stage of normal cellular involution. In the respira of epithelium, high columnar and low cuboidal, were found. The finding of well-differentiated and poorly differentiated tory epithelium, differentiated columnar cells normally undergo involution and are shed. Occasional columnar cells with ex zones in expiants exposed to benz(m,n,o)fluoranthene (in con tensive autophagic inclusions may have represented senescent trast to the occurrence of individual, abnormal cells among differentiated cells and not adverse effects of cultivation or otherwise normal cells) is important for two reasons. First, it treatment with hydrocarbons. illustrates a pitfall in analyses made in one experiment. In Endoplasmic Reticulum. Maintenance of rough endoplasmic several parallel histologie studies, this compound did not sup reticulum varied with the hydrocarbons used. In chrysene-, press differentiation nor alter proliferative activity. Second, the perinaphthoxanthene-, phenanthrene-, and benzo(a)fluorene- finding of an apparent biologic effect of any test agent in organ treatecl expiants, the endoplasmic reticulum was abundant and culture requires close attention to abnormalities produced by consisted of many cisterna! elements in longitudinal arrays culturing. The variations described for benz(m,n,o)fluoranthene (Figs. 5,6). It was less abundant after exposure to perylene, but are within the range observed in nutritionally deprived zones somewhat disorganized and not very abundant in benzo(a)py- of control expiants. These variations are significantly less rene-treated tracheas. After benz(m,n,o)fluoranthene treatment severe and specific than the alterations produced by the car the amount was variable; the state of the endoplasmic re cinogenic hydrocarbons. Alterations produced by F-DMBA, particularly at higher ticulum varied depending on whether the epithelium had maintained its columnar appearance. concentrations, were very extensive. At 50 /Â¿g/ml,columnar F-DMBA was a special case. At a low concentration (5 cells were transformed into flattened or squamous cells having /Â¿g/ml),many elements of endoplasmic reticulum were present, little resemblance to those in normal respiratory epithelium. The epithelium appeared stratified; complex cell interdigita- but at higher concentrations (25 and 50 ftg/ml), the endo tions occurred at all levels (Figs. 8, 9, 11), and desmosomes, plasmic reticulum was considerably depleted, and the cytoplasm was filled with smooth-membraned vesicles, possibly derived typical of stratified squamous epithelium (33, 35), connected the cells (Fig. 14). from the endoplasmic reticulum (Figs. 8, 9). The carcinogen benzo(a)pyrene produced variable results: The carcinogenic hydrocarbon benzo(a)pyrene produced in some expiants, very high columnar cells were maintained variable results. Where the epithelium maintained its columnar while, in others, stratified, rounded cells were produced. When appearance, endoplasmic reticulum cisternae were present; but this occurred, the cells were joined by complex cellular inter- in the squamous-appearing cells, much of the endoplasmic digitations. In the presence of 2.5 to 5 /tg/ml of DMBA and reticulum was lost and many free ribosomes were seen in the 50 jug/ml of benzo(a)pyrene, such major morphologic changes cytoplasm. In the presence of DMBA and benz(a)anthracene,

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1968 American Association for Cancer Research. Alterations of Rat TrachÃ©alEpithelium similar results were obtained (Figs. 16, 17, 25, 26). In some matrix in which ribosomes, myelin-like lamellae, and vacuoles, instances, however, a few cells retained portions of endoplasmic themselves membrane-bound, were sequestered (Fig. 28). reticulum, or there were cytoplasmic areas surrounded by cir Frequently, lysosomes were enlarged by coalescence or by cular profiles of endoplasmic reticulum (Figs. 25, 28). successive cycles of progressively extensive autophagy, par Golgi cisternae were present in most of the hydrocarbon- ticularly in the carcinogen-treated expiants. Examples can be treated expiants. Although their morphology and position in the seen in Figs. 8, 19, and 20. These autophagic vacuoles were cell varied considerably, even in in vivo material, some gener membrane bound and included vacuoles of various sizes as well alizations can be made. In those expiants treated with noncar- as ribosomes, glycogen, and dense bodies. Some of these in cinogenic hydrocarbons, without loss of endoplasmic reticulum, clusions were so complex and enlarged that they comprised a Golgi cisternae were often seen associated with endoplasmic large portion of the cell (Fig. 20). Since a study of large reticulum (Figs. 5, 6). In expiants treated with carcinogenic numbers of sections of multiple cell profiles did not produce hydrocarbons, leading to loss of endoplasmic reticulum, Golgi lateral interdigitations of such size, it was assumed that these cisternae were usually not apparent. When cells retained some inclusions were not sections through adjacent, protruding cells elements of endoplasmic reticulum, however, Golgi cisternae but were true inclusions within cells. Inclusions included mito were often found in close association. chondria, endoplasmic reticulum, ribosomes, and other cyto Lysosomal Derivatives (Autophagic Vacuoles, Residual plasmic components encompassed by a single membrane not Bodies, Dense Bodies, and Phagosomes). Throughout this sec wholly within the electron photomicrograph. Fig. 20 also shows tion the terminology used is that of de Duve and Wattiaux four structures of electron-dense granular material. It is pos (18). sible that, because of their surrounding membranes, these In control cultures, autophagic vacuoles were present in rela granules might represent portions of a lobated nucleus; how tively greater numbers and complexity than in respiratory ever, their internal material did not possess the characteristics epithelium in vivo. These are membrane-lined structures which of nuclear chromatin. contain some morphologically recognizable cytoplasmic com Phagosomes, which are protein adsorption droplets including ponents, indicating that the cell had sequestered portions of any phagocytic or pinocytic vacuoles in the cell (18), were not its own cytoplasm. Residual bodies were frequently found in particularly abundant in the control or the noncarcinogen hy control cultures (Fig. 2). These are membrane-lined inclusions drocarbon-treated expiants. However, at high concentrations of indigestible residues, such as membrane fragments or other of F-DMBA, many small vacuoles contained mucous or pro- components, which appear as whorls or myelin-like lamellae. teinaceous material probably derived from the lumen (Fig. 13). In the presence of the noncarcinogenic hydrocarbons, the In the presence of the carcinogenic hydrocarbons, such pino lysosomal derivatives increased in number as well as in internal cytic vacuoles pinched off from the cell membrane (Fig. 26). complexity. Some were seen only once. A circle of internalized It is possible that these vacuoles coalesced with others to pro cilia, for example, was encountered in a cell of a phenanthrene- duce the large mucous droplets frequently seen near the cell treated explant. A very large portion of the cell membrane, membrane. containing many cilia, had pinched off completely, thus forming In carcinogen-treated expiants, internalized cilia were found a large internal vacuole. The cilia, arranged in a circle, pro occasionally. In a benzo(a)pyrene-treated expiant, for example, truded into the vacuolar lumen, while the basal bodies re a cilium surrounded by its cell membrane sheath had been mained within the cytoplasm. Similar structures have been pulled deep into the cytoplasm, but it maintained its charac seen in rat nasal epithelium (54). teristic structure. Such examples were also found in bcnz(a)an The greatest number and complexity of lysosomal derivatives were demonstrated in the F-DMBA-treated tissues. Lipid thracene-treated explants. Intracytoplasmic flagella have fre droplets with internalized vacuoles, as well as autophagic quently been found in human skin (63), in fibroblasts from a vacuoles (Fig. 8) and residual bodies (Figs. 9, 15) were found. human brain tumor (47), and in human anaplastic bronchial The residual body in Fig. 15 is a good example of this type of carcinoma (55). lysosomal derivative. The fine, particulate matrix contains a Cytoplasmic Filaments. Cytoplasmic filaments were not series of myelin-like lamellae, and the structure is surrounded found in the columnar cells of expiants treated with noncar by a single limiting membrane. Autophagic vacuoles in which cinogenic hydrocarbons or in controls, but they were frequently mitochondria were clearly evident were also frequently en observed in basal cells, particularly near the basement mem countered (Fig. 11). Many small vacuoles, possibly remnants brane. of endoplasmic reticulum, were usually present within these In the case of F-DMBA, little filamentous material was autophagic vacuoles. found at the lower concentration used, but at the higher con Similar and even more complex structures were present in centrations filaments were present at all levels of the epithe other carcinogen-treated expiants. Autophagic vacuoles with lium: in cells near the lumen (Fig. 12), at the center of the several mitochondria and various other cytoplasmic sequestra epithelium (Fig. 11), and connected to typical desmosomes (Fig. tions were observed in most of the expiants (Fig. 17). Unusual 14). None of the cells containing such filaments were columnar, lysosomal derivatives of intermediate morphology, consisting nor were they identifiable entirely as basal cells. partly of multivesicular and partly of dense or residual bodies With benzo(a)pyrene, those cells retaining a columnar ap surrounded by unit membranes, were occasionally found (Fig. pearance had parallel arrays of endoplasmic reticulum, while 24). Other intermediate inclusions contained a fine granular the cytoplasm of the cuboidal or squamous cells had little endo-

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1968 American Association for Cancer Research. E. Roter Dirksen and T. Timothy Crocker plasmic reticulum and a considerable amount of filamentous mitochondrial alterations, and formation of cytoplasmic se components. questrations and lysosomal derivatives, to name a few. Mor In the case of both benz(a)anthracene and DMBA, none of phologic alterations in cells appear, then, to be nonspecific, but the various cytoplasmic changes were more dramatic than the this may be due to the lack of versatility of cells in responding appearance of massive amounts of filamentous material at all to a variety of agents rather than to the lack of specific action levels of the epithelium, for example, in parallel arrays and, of those agents. The intermediate molecular pathways may in cross section, in cells near the lumen (Figs. 17, 25, 26) and differ for each agent or stimulus, but the ultimate morphologic at other levels (Figs. 16, 22). In many expiants such material changes are apparently the same regardless of the causative was often associated with lipid droplets (Fig. 18). agents. In the present experiments, disruptive cellular changes There was also a correlation between the decrease or absence were qualitatively similar but were quantitatively less exten of parallel arrays of endoplasmic reticulum and the increased sive in control expiants or in expiants treated with weakly amounts of cytoplasmic filaments found in a particular cell. carcinogenic or noncarcinogenic hydrocarbons than in expiants Furthermore, those cells retaining their columnar appearance treated with the benz(a)anthracenes, including F-DMBA or contained few, if any, cytoplasmic filaments. These correla benzo(a)pyrene. The latter compounds also produced an epi tions do not apply, however, to filamentous material in cells thelial state and a cell type in respiratory epithelium which near the basement membrane where such material was normally was unique to these hydrocarbons. Columnar cell differentia found. Finally, these cytoplasmic filaments were similar in tion was replaced by stratification of rounded cells having in structure and distribution to those observed in stratified squa- tercellular digitations, desmosomes, intracytoplasmic filaments, mous epithelium (33). and aberrant mitochondrial cristae, with the result that rat Mitochondria. In the presence of most of the noncarcinogenic respiratory epithelium became virtually identical with that hydrocarbons, the mitochondria maintained their characteristic described for human bronchial carcinoma in situ (61) and re size and shape (Fig. 5). After treatment with F-DMBA, how sembled, in part, a human anaplastic bronchial carcinoma ever, the mitochondria were often bizarrely shaped (Fig. 10), (55). It is significant that polycyclic hydrocarbons of classes but normal mitochondria were also found in these expiants. which are carcinogenic for rodents produced this morphologic Because of the naturall}" pleomorphic nature of these organelles, imitation of bronchogenic carcinoma; it is also noteworthy only extremely aberrant deviations were considered abnormal. that the most potent carcinogen DMBA was active at much Deformed mitochondria with longitudinal cristae were fre lower concentrations than the weakly carcinogenic benz(a)an- quently seen in the expiants exposed to benz(a)anthracene thracene and F-DMBA. and DMBA (Figs. 22, 23) but were rarely observed with benzo(a)pyrene. Endoplasmic Reticulum Alterations Within lytic and residual bodies, mitochondria were often The most characteristic and general alteration produced by found associated with other eytoplasmic components (Figs. 11, biologically effective concentrations of F-DMBA, DMBA, 17) or with filamentous material (Fig. 10). benz(a)anthracene, and benzo(a)pyrene was the considerable The Nucleolus. The great variability in size, shape, and loss of parallel arrays of endoplasmic reticulum. Similar altera structure of both nuclei and nucleoli makes it difficult to tions have been found in the Morris 5123 and particularly in interpret many of the changes which occurred in these or the Dunning LC/18 hepatomas (41). In the fast-growing Dun ganelles. The number of nucleoli appeared to increase par ning LC/18 hepatoma, parallel profiles of endoplasmic reticulum ticularly in benz(a)anthracene- and DMBA-treated expiants cisternae were rare; the cytoplasm was full of vesicles and (Fig. 21). These nucleoli often appeared as if they were frag free ribosomes. A fine structural comparison of hepatomas with mented, and they contained multiple, electron-dense, granular widely varying growth rates further demonstrated that fast- inclusions. Besides the fragmentation of the nucleoli into diffuse growing hepatomas have a simply organized cytoplasm (17, structures, the nucleonema itself became dispersed and lost 26). In the Iteuber H-35 hepatoma the endoplasmic reticulum much of its normal architecture. In addition, electron-dense was composed of strands coursing among mitochondria and material appeared in clumped configurations throughout the not of the parallel arrays so characteristic of liver cells (36). nucleolus. Such morphology was also observed in control ex- Studies on the early effects of 3'-methyl-4-dimethylaminoazo- plants (Fig. 1), but more frequently in the carcinogenic hydro benzene on rat liver (40) demonstrated that the most striking carbon-treated expiants. Nucleoli in explants treated with non- change occurred in the endoplasmic reticulum, which was no carcinogenic hydrocarbons were similar morphologically to longer present in parallel arrays but was scattered and dis those found in control expiants. organized. Free ribosomes, annulate lamellae, and a dispersed Nuclear volume increased in expiants treated with carci endoplasmic reticulum seemed to be related to the high cell nogenic hydrocarbons, particularly in the squamous cells (Fig. division of hepatomas. The more differentiated cytoplasm of 21). Irregularly shaped nuclei were often encountered, and slow-growing hepatomas with their extensive rough and smooth various types of nuclear inclusions were not rare (Fig. 8). endoplasmic reticulum suggested that the degree of organiza tion of these cytoplasmic organelles varied with rate of growth. DISCUSSION The effects of the liver carcinogens dimethylnitrosamine and thioacetamide on the fine-structural changes of rat liver have Cells of many types respond to a wide spectrum of stimuli been thoroughly studied (5, 20). Although other cytoplasmic in a similar manner: loss of organized endoplasmic reticulum, organelles retained their typical morphology after dimethyl-

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1968 American Association for Cancer Research. Alterations of Rat TrachÃ©alEpithelium nitrosamine administration, the rough endoplasmic reticulum in mouse pulmonary adenomas induced with methylcholanthrene or urethan (57). These cytoplasmic "lakes," consisting became swollen and fragmented. Small vesicles with detached particles became abundant, resembling smooth endoplasmic of dense particles in a fairly homogeneous matrix, were often reticulum. Studies by these authors on isolated microsomes continuous with cisternae of the endoplasmic reticulum. Such and ribosomes (5) further demonstrated that dimethylnitro- cytoplasmic "lakes" were frequently found in the expiants samine affects the structural relationship between ribosomes treated with carcinogenic hydrocarbons in our studies (Figs. and membranes as well as the membranes themselves. 25, 28). Evidence for ultrastructural changes in the endoplasmic Allison and coworkers (1, 2) have hypothesized that lyso- reticulum of spontaneously occurring human cancer has also somes are involved in polycyclic aromatic hydrocarbon car- been demonstrated. Many free ribosomes with little organized cinogenesis, based on evidence from combined fluorescence endoplasmic reticulum, as well as variations in mitochondria! microscopy and histochemistry that the carcinogenic hydrocar structure and changes in the position and size of the Golgi bons were concentrated in the lysosomes. They conjectured that complex, occurred in human prostatic carcinoma (32). A these hydrocarbons induced permeability changes in the lyso recent electron microscope study (61) showed that cells of a somal membranes, allowing the release of lysosomal enzymes human epidermoid bronchial carcinoma in situ (preinvasive which produced secondary effects (possibly involving RNA or neoplasm) had become primitive in appearance, with loss of DNA) perpetuated after mitosis. Their results (1) are of in organized endoplasmic reticulum and increased free cytoplas- terest in relation to ours, since the increased presence of lyso mic ribosomes. The cytoplasm of all the cells was filled with somal derivatives in our material corresponds to the carcino- masses of cytoplasmic filaments, while large intercellular spaces genicity of the hydrocarbons used. Their data (1) do not dif occurred between the neoplastic epithelial cells from the lumen ferentiate between the two ways in which hydrocarbons could to the basement membrane. be concentrated in lysosomes, i.e., exophagy (pinocytic vac That disruption of the organized endoplasmic reticulum is uoles) or endophagy (autophagic vacuoles). Lysosomes, there commonly an early response to cellular injury has been dem fore, need not be the site of action of carcinogenic hydrocar onstrated by the administration of certain antimetabolites and bons but could be receptors for carcinogen-injured cytoplasmic by some deficiency states. However, such changes also occur as components, or they might be both. Weissman et al. (62) re stable morphologic features of fast-growing hepatomas induced ported that isolated lysosomes are not disrupted by the tumor- by carcinogens, other carcinogen-induced tumors, and spon initiating polycyclic hydrocarbons methylcholanthrene and taneous tumors. The meaning of these alterations in relation DMBA, although croton oil or its more active tumor-promoting to the carcinogenic process is not clear at this time. In the fractions are lytic. Since lysosomal instability as tested in this present study, however, the greater degree of disruption by the fashion was not increased by certain carcinogenic hydrocar carcinogenic benz(a)anthracenes, including F-DMBA and bons, an alternative explanation for the presence of these hy benzo(a)pyrene, and the lack of effect of the noncarcinogenic drocarbons in lysosomes may be considered. hydrocarbons indicate that these two groups of compounds Since cellular autophagy occurs in physiologic and pathologic differ significantly in capacity to alter the endoplasmie retic cytoplasmic reabsorption, the large number of autophagic vac ulum. Since the group producing changes in the endoplasmic uoles and residual bodies observed in our carcinogen-treated reticulum includes known carcinogens, of which two are potent expiants may represent involution of these cells, hastened by tumor initiators, these early effects may be related to carci- biologically active polycyclic hydrocarbons. In this context, it nogenesis. is interesting to note that those cells most completely modified to a squamous type, with cytoplasm containing large amounts Lysosomes and Lysosomal Derivatives of filamentous material, have few lysosomal derivatives (e.g., There is electron microscopic evidence for the presence of Figs. 12, 16). Hence they may be formerly differentiated cells complex laminated and residual bodies in DMBA-induced rat which have completed the reabsorption and degradation of cytoplasmic organelles, leaving the cytoplasm "empty"; or sarcomas (37), of cytoplasmic sequestrations in methylcholanthrene-induced pulmonary adenomas in the mouse (57), and they may be basal cells modified to the squamous state, in of these and other lysosomal derivatives in fast-growing hepa which lysosomes are less common and filaments are increased. tomas (26). However, such cellular autophagy also has been Such cells may be unable to differentiate into columnar cells observed under normal conditions (21), being enhanced in cells in the presence of carcinogenic hydrocarbons. Squamous cells undergoing involution during morphogenesis (cf. 18). Focal produced in this way may be as uniquely differentiated as cytoplasmic degradation (25, 28, 58) has been demonstrated columnar cells and are able to survive in the presence of these in many cell types and may possibly serve as a mechanism for hydrocarbons but have no capacity to assume a columnar state. the bulk segregation and digestion of portions of the cytoplasm When the respiratory epithelium reaches the state of stratified by the cell. Specific metabolic inhibitors (e.g., /8-3-thienyla- squamous differentiation, it closely resembles that described lanine, an analog of phenylalanine; azaserine, a purine biosyn by Watson in bronchogenic carcinoma in situ (61). Reversibility thesis inhibitor; and triparanol, a cholesterol inhibitor), star of this state in the respiratory epithelium after removal of the vation, and castration produced cytoplasmic sequestrations carcinogen has been reported in organ culture (16) and in vivo which were subsequently degraded (27, 56, 58). in dogs (59) and man (3). The ultrastructure of the squamous Segregation of cytoplasmic components by membranes, pos cells in the present study is apparently identical to that of sibly derived from the endoplasmic reticulum, was demonstrated similar cells in preinvasive squamous cell carcinoma (61).

MAY 1968 911

However, reversibility indicates that these ultrastructural de carcinosarcomas (50, 51). Although these alterations differed tails need not coincide with neoplastic transformation. They in details, the final change was a general disorganization and may be regarded as structural features imposed on the respira fragmentation of the nucleonema with loss of typical nucleolar tory epithelium by hydrocarbons or other agents acting to structure, as in proflavin-treated cells or with large, dense delete mechanisms or structures which are expressed in the granules, as in Walker 256 carcinosarcoma. It may be that columnar state of differentiation. The site of action of agents intranuclear effects need not occur in carcinogenic transforma having this effect could be limited to the cytoplasm and may tion but that, as suggested (39), disruptions in metabolic reg not involve the genome; however intranuclear sites of action ulatory circuits (presumably by cytoplasmic effects) could be should also be taken into consideration. involved. It is possible, of course, that the nucleus is affected at the molecular level, and that the changes cannot be per Mitochondrial Changes ceived at the level of resolution attained. Although there is Mitochondrial changes, although not extensive, were encoun biochemical evidence for the binding of hydrocarbon carci tered particularly in F-DMBA-, DMBA-, and benz(a)anthra- nogens to both DNA and RNA (4, 12, 13, 43) as well as to cene-treated trachÃ©alexplants. These alterations usually con protein (24, 34, 42), the significance of this binding remains sisted of abnormally positioned cristae or of aberrantly shaped unclear. mitochondria. In methylcholanthrene-induced carcinoma of the mouse cervix, the mitochondria of the epithelial cells contained ACKNOWLEDGMENTS intramitochondrial dense bodies (46). Similar mitochondrial alterations were produced in mouse epidermis after local appli The authors wish to thank Mrs. Kathleen Arras and Miss Emma cation of carcinogens (49). Some authors have suggested, how Jean Battles for their valued and competent technical assistance ever, that these mitochondrial changes may merely represent a in electron microscopy. Organ cultures and histologie preparations nonspecific cytotoxic effect (36, 46). were performed with the expert assistance of Beryl J. Nielsen and Of considerable interest is the finding (10, 11), in adenovirus- Susan A. Wells. 12-induced hamster tumors, of anomalous, bell-shaped mito chondria lacking most of their cristae, persisting after as many REFERENCES as 28 passages in culture. The persistence of these mitochondrial aberrations excludes the possibility that such modifications were 1. Allison, A. C., and Mallucci, L. Uptake of Hydrocarbon Car responses to conditions of culture or that they were artifacts. cinogens by Lysosomes. Nature, 203: 1024-1027, 1964. The noncarcinogenic dye 2-methyl-4-dimethylaminoazoben- 2. Allison, A. C., and PatÃ³n, G. R. Chromosome Damage in Hu zene duplicates the morphologic alterations produced by other man Diploid Cells Following Activation of Lysosomal En hepatotoxins, some of which, such as 3'-methyl-4-dimethyl- zymes. Nature, 207: 1170-1173, 1965. aminoazobenzene, are carcinogenic (30). Mitochondria show 3. Auerbach, D., Stout, A. P., Hammond, E. C., and Garfinkel, L. Changes in Bronchial Epithelium in Relation to Sex, Age, Res a pronounced response to this drug, both in population increase idence, Smoking and Pneumonia. New Engl. J. Med., 267: and in morphology. Transverse double membranes, as well as 111-119, 1962. arrays of long, cristae-like lamellae, are often found within 4. Ball, J. K., McCarter, J. A., and Smith, M. F. The Interaction these mitochondria. Drastic alterations in mitochondrial mor in vitro of Polycyclic Aromatic Hydrocarbons with Deoxyribo- phology likewise have been seen in human renal clear-cell car nucleic Acids. Biochim. Biophys. Acta, 103: 275-285, 1965. cinoma (48). The abnormality most frequently encountered 5. Benedetti, E. L., and Emmelot, P. Effect of Dimethylnitrosa- is the presence of lamellar structures and concentric arrays of mine on the Endoplasmic Reticulum of Rat Liver Cells. Lab. paired membranes resembling the parallel layering of elongated Invest., 15: 209-216, 1966. cristae. Such alterations have been observed under various 6. Bennett, H. S., and Luft, J. H. s-Collidine as a Basis for Buffer other experimental conditions. See Trump and Ericsson (60) ing Fixatives. J. Biophys. Biochem. Cytol., 6: 113-114, 1959. for a review of this problem. 7. Bergmann, E. D., Blum, J., and Haddow, A. Carcinogenic Ac tivity of Some Fluorinated Polycyclics. Nature, 200: 480, 1963. 8. Bernhard, W., Frayssinet, C., Lafarge, C., and Le Breton, E. Nucleolar Lesions LÃ©sionsNuclÃ©olaires PrÃ©coces ProvoquÃ©es par L'Aflatoxine In the present experiments with trachÃ©alepithelium, exten dans les Cellules HÃ©patiquesdu Rat. Compt. Rend. Acad. Sci., sive ultrastructural changes were not often found in the 261: 1785-1788, 1965. nucleoli, either with the noncarcinogenic or carcinogenic hydro 9. Bernhard, W., and Granboulan, N. The Fine Structure of the carbons, with the exception of benz(a)anthracene and, to a Cancer Cell Nucleus. Exptl. Cell Res., Suppl., 9: 19-53, 1963. lesser extent, DMBA. In general, the major response occurred 10. Bernhard, W., and Tournier, P. Modification persistante des mitochondries dans des Cellules tumorales de hamster trans in the cytoplasm. Aside from an increased nuclear/cytoplasmic formÃ©espar 1'adenovirus 12. Intern. J. Cancer, /: 61-80, 1966. ratio and nucleolar disorganization, few alterations could be 11. Bernhard, W., Tournier, P., and Lorans, G. Anomalies Mito- correlated with the properties of the hydrocarbons used. chondriales Persistantes dans les Cellules Tumorales de Ham However, the nucleus and nucleolus are likely organdÃesfor ster Induites par 1'Adenovirus 12. Compt. Rend. Acad. Sci., carcinogenic interaction. Alterations in nucleolar fine structure Ã•61:2137-2140, 1965. have been encountered as a response to aflatoxin (8), methyl- 12. Boj-land, E., Green, B., and Liu, S. L. Factors Influencing the cholanthrene (9), proflavin (52), actinomycin (53), in cells of Interaction of Polycyclic Hydrocarbons and DNA. Biochim. a cultured gastric epithelioma (64), and in the Walker 256 Biophys. Acta, 87: 653-663, 1964.

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13. Brookes, P. Quantitative Aspects of the Reaction of Some 31. Luft, J. H. Improvements in Epoxy Resin Embedding Meth Carcinogens with Nucleic Acids and the Possible Significance ods. J. Biophys. Biochem. Cytol., 9: 409-414, 1961. of Such Reactions in the Process of Carcinogenesis. Cancer 32. Mao, P., Nakao, K., and Angrist, A. Human Prostatic Carci Res., 26: 1994-2003, 1966. noma: An Electron Microscope Study. Cancer Res., 26: 955- 14. Crocker, T. T., and Nielsen, B. I. Effect of Carcinogenic Hy 973, 1966. drocarbons on Suckling Rat Trachea in Living Animals and in 33. Mercer, E. H. Keratin and Keratinization. New York: Per- Organ Cultures. In: L. Severi (ed.), Lung Tumours in Animals, gamon Press, 1961. Third Quadrennial Intern. Conf. Cancer, University of Perugia, 34. Miller, J. A., and Miller, E. C. A Survey of Molecular Aspects June 24-29, 1965, Vol. 3, pp. 765-787. University of Perugia, of Chemical Carcinogenesis. Lab. Invest., 16: 217-241, 1966. Italy : Division of Cancer Research, 1966. 35. Nakai, T., Shubik, P., and Feldman, R. An Electron Micro 15. Crocker, T. T., and Nielsen, B. I. Specificity of Effects of scopic Study of Skin Carcinogenesis in the Mouse with Special Carcinogenic and Noncarcinogenic Hydrocarbons on Respira Reference to the Intramitochondrial Body. Exptl. Cell Res., tory Epithelium. Ninth Intern. Cancer Congr., October 23-29, 27: 608-611, 1962. 1966. 36. Novikoff, A. B., and Essner, E. Pathological Changes in Cyto 16. Crocker, T. T., Nielsen, B. I., and Lasnitzki, I. Carcinogenic plasmic Organelles. Federation Proc., 21: 1130-1142, 1962. Hydrocarbons. Arch. Environ. Health, 10: 240-250, 1965. 37. Parry, E. W., and Ghadially, F. N. Ultrastructure of Car 17. Dalton, A. J. An Electron Microscopical Study of a Series of cinogen-induced Rat Sarcoma. Cancer, IS: 1026-1040, 1965. Chemically Induced Hepatomas. In: P. Emmelot and 0. Muhl- 38. Parsons, D. F. A Simple Method for Obtaining Increased Con bock (eds.), Cellular Control Mechanisms and Cancer, pp. 211- trast in Araldite Sections by Using Post-fixation Staining of 225. New York: Elsevier Publishing Co., 1964. Tissues with Potassium Permanganate. J Cell Biol., 11: 492- 18. de Duve, C., and Wattiaux, R. Functions of Lysosomes. In: 497, 1961. V. E. Hall (ed.), Annual Review of Physiology, Vol. 28, pp. 39. Pitot, H. C., and Heidelberger, C. Metabolic Regulatory Cir 435-492. Palo Alto, California: Annual Reviews, Inc., 1966. cuits and Carcinogenesis. Cancer Res., 23: 1694-1700, 1963. 19. Dirksen, E. R., and Crocker, T. T. Centriole Replication in 40. Porter, K. R., and Bruni, C. An Electron Microscope Study of the Early Effects of 3'-Me-DAB on Rat Liver Cells. Cancer Differentiating Ciliated Cells of Mammalian Respiratory Epi thelium. An Electron Microscopic Study. J. Microscopie, 6: Res., 19: 997-1010, 1959. 629-644, 1966. 41. Porter, K. R., and Bruni, C. Comparative Fine Structure of 20. Emmelot, P., and Benedetti, E. L. Some Observations on the Slow- and Fast-Growing Hepatomas. Acta UniÃ³ Intern. Contra Effect of Liver Carcinogens on the Fine Structure and Func Cancrum, 20: 1271-1274, 1964. tion of the Endoplasmic Reticulum of Rat Liver Cells. In: 42. Pullman, A. The Theory' of Chemical Carcinogenesis and the R. J. C. Harris (ed.), Protein Biosynthesis, pp. 99-123. New Problem of Hydrocarbon-protein Interactions. Biopolymers, York : Academic Press, 1961. Symp., 1: 47-65, 1964. 21. Ericsson, J. L. E., Trump, B. F., and Weibel, J. Electron Mi 43. Pullman, B. Electronic Aspects of the Interactions Between croscopic Studies of the Proximal Tubule of the Rat Kidney. the Carcinogens and Possible Cellular Sites of Their Activity. II. Cytosegresomes and Cytosomes: Their Relationship to J. Cellular Comp. Physiol., 64: 91-109, 1964. Each Other and to the Lysosome Concept. Lab. Invest., 14: 44. Reynolds, E. S. The Use of Lead Citrate at High pH as an 1341-1365, 1965. Electron-opaque Stain in Electron Microscopy. J. Cell. Biol., 22. Falk, H. L., Kotin, P., and Thompson, S. Inhibition of Car 17: 208-212, 1963. cinogenesis. Arch. Environ. Health, 9: 169-179, 1964. 45. Rhodin, J., and Dalhamn, T. Electron Microscopy of the 23. Farquhar, M. G., and Palade, G. E. Junctional Complexes in TrachÃ©alCiliated Mucosa in Rat. Z. Zellforsch. Mikroskop. Various Epithelia. J. Cell Biol., 17: 375-412, 1963. Anat., 44: 345-412, 1956. 24. Heidelberger, C. Studies on the Molecular Mechanism of Hy 46. Schrodt, G. R., and Foreman, C. D. Methylcholanthrene- drocarbon Carcinogenesis. J. Cellular Comp. Physiol., 64: 129- induced Carcinoma of the Mouse Cervix: An Electron Micro 148, 1964. scope Study. Cancer Res., 25: 802-811, 1965. 25. Hruban, Z., Spargo, B., Swift, H., Wissler, R. W., and Klein 47. Schuster, F. L. Ciliated Fibroblasts from a Human Brain Tu feld, R. G. Focal Cytoplasmic Degradation. Am. J. Pathol., mor. Anat. Record, 150: 417-421, 1964. 42: 657-684, 1963. 48. Seljelid, R., and Ericsson, J. L. E. An Electron Microscopic 26. Hruban, Z., Swift, H., and Rechcigl, M., Jr. Fine Structure of Study of Mitochondria in Renal Clear Cell Carcinoma. J. Transplantable Hepatomas of the Rat. J. Nati. Cancer Inst., 35: 459-495, 1965. Microscopie, 4: 759-770, 1965. 27. Hruban, Z., Swift, H., and Slesers, A. Effect of Azaserine on 49. SetÃ¤lÃ¤,K.,Merenmies, L., Niskanaen, E. E., Nyholm, M., and the Fine Structure of the Liver and Pancreatic Acinar Cells. Stjernvall, L. Mechanism of Experimental Tumorigenesis. VI. Cancer Res., 25: 708-723, 1965. Ultrastructural Alterations in Mouse Epidermis Caused by 28. Hruban, Z., Swift, H., and Wissler, R. W. Analog-induced In Locally Applied Carcinogen and Dipole-type Tumor Promoter. clusions in Pancreatic Acinar Cells. J. Ultrastruct. Res., 7: J. Nati. Cancer Inst., 26: 1155-1190, 1960. 273-285, 1962. 50. Shankarnarayan, K., and Busch, H. Dense Granules in Nu 29. Klein, M. Susceptibility of Strain B6AF/J Hybrid Infant Mice cleoli of Walker Carcinosarcoma Cells of the Rat. Exptl. Cell to Tumorigenesis with 1,2-Benzanthracene, Deoxycholic Acid, Res., 38: 434-437, 1965. and 3-methylcholanthrene. Cancer Res., S3: 1701-1707, 1963. 51. Shankarnarayan, K., Muramatsu, M., Smetana, K., and Busch, 30. Lafontaine, J. G., and Allard, C. A Light and Electron Micro H. Ultrastructural Studies on RNA and DNA Components of scope Study of the Morphological Changes Induced in Rat Isolated Nucleoli of Walker 256 Carcinosarcoma. Exptl. Cell Liver Cells by the Azo Dye 2-Me-DAB. J. Cell Biol., %Z: 143- Res., 41: 81-98, 1966. 172, 1964. 52. Simard, R. Specific Nuclear and Nucleolar Ultrastructural Le-

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sions Induced by Proflavin and Similarly Acting Antimetabo- mous Metaplasia Produced in Dogs by Cigarette Smoke lites in Tissue Culture. Cancer Res., 36: 2316-2328, 1966. Condensate. J. Nati. Cancer Inst., S3: 487-495, 1964. 53. Smuckler, E. A., and Benditi, E. P. The Early Effects of Ac- 60. Trump, B. F., and Ericsson, J. L. E. Some Ultrastructural and tinomycin on Rat Liver: Changes in Ribosomes and Poly- Biochemical Consequences of Cell Injury. In: B. W. Zweifad, somes. Lab. Invest., 14: 1699-1709, 1965. L. Grant and R. T. McCluskey (eds.), The Inflammatory Proc 54. Stockinger, L. Die Ultrastruktur des Flimmerepithels des ess, pp. 35-120. New York: Academic Press, Inc., 1965. Nasenseptums der Ratte. Z. Zellforsch. Mikroskop, Anat. 59: 61. Watson, J. H. L., Bryant, V., and Brinkman, G. L. The Ultra- 443-466, 1963. structure of Epithelium of Human Bronchus in Epidermoid 55. Stoebner, P., Cussac, Y., Porte, A. and Le Gal, Y. Ultrastruc Carcinoma. In: R. Uyeda (ed.), Electron Microscopy 1966. ture of Anaplastia Bronchial Carcinomas. Cancer, ÃªO: 286- Proc. Sixth Intern. Congr. Electron Microscopy, Vol. II, pp. 294, 1967. 759-760. Tokyo: Maruzen Company, Ltd., 1966. 56. Svoboda, D. J. Ultrastructural Changes Produced by Protein 62. Weissman, G., Troll, W., Van Duuren, B., and Sessa, G. A New Related Deficiencies in the Rat Liver. Am. J. Pathol., Ifi: Action of Tumor-promoting Agents: Lysis of Cell and Intra- 353-379, 1964. cellular Membranes. J. Clin. Invest., 46: 1131, 1967. 57. Svoboda, D. J. Electron Microscopic Study and Murine Pul 63. Wilson, R. B., and McWhorter, A. Isolated Flagella in Human monary Adenomas. Acta UniÃ³ Intern. Contra Cancrum, 20: Skin. Lab. Invest., 13: 242-249, 1963. 1331-1336, 1964. 64. Wolff, E., Zagury, D., and Leger, L. Recherches sur les Condi 58. Swift, H., and Hruban, Z. Focal Degradation as Biological tions de la Culture Organotypique de Cancer Humains. II. Process. Federation Proc., 23: 1026-1037, 1964. Etude au Microscope Electronique. Presse Med., 56: 2759- 59. Tipton, D. L., and Crocker, T. T. Duration of Bronchial Squa- 2762, 1962.

Fig. 1-3. Acetone controls. Fig. 1. Low power view of the trachÃ©alepithelium after 11 days in culture. Examples of ciliated (C), goblet (G), brush (Br), and basal (B) cells are seen. Microvilli (mv) are present at the borders of brush cells and are interspersed between cilia (c). Large inter cellular spaces (i) can be found near the basement membrane (bm) and surrounding the basal cells (B), but not near the lumen (L). The nuclei (N) are present at the lower portion of the cell; in several of them, nucleoli (ne) are seen. Fibroblasts (/) and elastin (el) are found underneath the basement membrane. X 5,000. Fig. 2. Portion of a goblet cell showing several Golgi cisternae (Gc), mucous droplets (md), and mitochondria (m). A residual body (rb) has a few myelin-like lamellae within it. This cell has maintained its normal morphology and is comparable to cells in non- cultured material. X 29,000. Fig. 3. Extensive stacks of endoplasmic reticulum can be seen in this goblet cell. X 31,000. Figs. 4-7. Noncarcinogens. Fig. 4. 6.7 Mg/ml chrysene. After 11 days, the columnar appearance of the epithelium was maintained. Several of the cells extend from the basement membrane (bm) to the lumen (L). X 5,000. Fig. 5. 20 fig/ml perinaphthoxanthene. Portion of a cell in which well-developed endoplasmic reticulum (er) and Golgi cisternae (Gc) with numerous vesicles can be seen. A mucous droplet (md) seems to be directly connected to elements of the endoplasmic reticulum. X 37,000. Fig. 6. 20 Mg/ml benzo(a)fluorene. Parts of two goblet cells separated by a cell membrane (cm) are seen here. Note the extensive endoplasmic reticulum (er), Golgi cisternae (Gc), mitochondria (m), and mucous droplets (md) with fragmentary boundary mem branes. Further evidence of morphologic normality is the close contact between these cells. X 8,000. Fig. 7. 6.7 Mg/ml chrysene. Cell turn-over with differentiation of new cells is inferred here from the presence of several centrioles in cross-section (cn^) and in longitudinal section (cnt) and by several basal bodies (b) at the cell surface. X 37,000. Figs. 8-15. F-DMBA. Fig. 8. 50 /ig/ml. The epithelium becomes low due to flattening of cells near the lumen (L), although evidence of some differentia tion is shown by the persistence of cilia (c). The endoplasmic reticulum consists primarily of fragmented, swollen vesicles (er). Many residual bodies (rb) and autophagic vacuoles (a) as well as intercellular spaces (i) are found in these expiants. Enlarged nucleoli (ne) and nuclear inclusions (as in N) are fairly frequent. Many fibroblasts (/) are seen below the basement membrane (bm). X 4,000. Fig. 9. 50 Mg/ml. Although much of the endoplasmic reticulum has been converted to smooth-surfaced vesicles (erf), some rough- surfaced elements (er,) are still present. A residual body (rb) with myelin-like lamellae is present. Notice the flattened appearance of the cells. X 8,000. Fig. 10. 50 Mg/ml. Portion of a cell which shows an abnormally shaped mitochondrion (m) surrounded by tufts of cytoplasmic fila ments (F). Notice the abundant free ribosomes in the cytoplasm. X 30,000. Fig. 11. 50 Mg/ml. Parts of several cells. Some have large bundles of cytoplasmic filaments (F), many vesicles of fragmented endo plasmic reticulum (ert), and complex intercellular connections (ci). A double-membraned autophagic vacuole (a) contains an undi gested mitochondrion and many vesicles. Multivesicular bodies (mb) with few or many small vesicles and with light or dark background matrices are present, x 14,000. Fig. 12. 50 Mg/ml. A cell near the lumen (L) with masses of cytoplasmic filaments (F) and little endoplasmic reticulum. Notice the flattened appearance and the undifferentiated state of the cell. X 44,000. Fig. 13. 5 Mg/ml. Although many cells maintained their normal morphology at this lower concentration, others contained little organ ized endoplasmic reticulum and many phagosomes (p) of undetermined origin. X 33,000. Fig. 14. 25 Mg/ml. Several desmosomes (oÃ)with bundles of filaments in cross-section (F) connect two cells, separated by their cell membranes (cm). X 33,000.

914 CANCER RESEARCH VOL. 28

Fig. 15. 50 Mg/ml. This residual body contains several stacks of myelin-like lamellae (my), and probably represents a final stage in the digestion of internalized cytoplasmic components. X 32,000. Figs. 16-18. DMBA. Fig. 16. 5 /xg/ml. Portions of two markedly altered cells near the lumen (L) are separated by cell membranes (arrows) studded with desmosomes (d). The flattened cell closest to the lumen is almost devoid of organelles while the cell below contains many bundles of cytoplasmic filaments (F), a dense body (db), and a few strands of endoplasma: reticulum (er). X 44,000. Fig. 17. 5 /ig/ml. In this cell few normal constituents are present. Extensive cytoplasmic filaments (F), and little endoplasmic retic ulum, are seen. An autophagic vacuole (a) with several mitochondrial profiles as well as two types of residual bodies (rbÂ¡-rbt) are also present. X 19,000. Fig. 18. 5 /ig/ml. Cytoplasmic filaments (F) are found near lipid droplets (id). A few short elements of endoplasmic reticulum (er) and many free ribosomes are seen in the cytoplasm. X 38,000. Figs. 19, 20. Benzo(a)pyrene. Fig. 19. 6 Mg/m'- A considerably enlarged nucleus (N) almost surrounds an enlarged autophagic vacuole (a) containing dense bodies (db), smooth-surfaced vesicles of endoplasmic reticulum (ert) as well as ribosomes and other membranous components. Parallel arrays of endoplasmic reticulum (ert) also can be seen in this cell. X 18,000. Fig. 20. 6 /ig/ml. An extremely engorged autophagic vacuole (not fully pictured) is delineated at several points by arrows. This gi gantic vacuole contains many mitochondria (m), mucous droplets (md), and elements of rough endoplasmic reticulum (er,) as well as smooth-surfaced vesicles (ert) ; possibly fragmented endoplasmic reticulum. The four structures (gt-gt) of van-ing sizes consist of electron-dense, particulate material adjoining an electron-lucent, amorphous substance. Many free ribosomes (r) also are seen. X 32,000. Figs. 21-28. Benz (a) anthracene. Fig. 21. 50 ng/mi. The trachea! epithelium has lost much of its columnar appearance and consists of stratified rounded cells. Cells are connected by complex interdigitations (ci), have little organized endoplasmic reticulum (er) and contain few organelles associated with differentiation. The basement membrane (bm) is extremely diffuse. X 8,000. Fig. 22. 50 /ig/ml. The mitochondria have cristae parallel to the outer longitudinal membrane (mÂ¡,mt, ms). Scarcely any endoplasmic reticulum, many free ribosomes (r), and much filamentous material (F) characterize the cytoplasm of these cells. Unit-membraned vacuoles (Vj-Vj) filled with amorphous, mucoid material are frequent. X 63,000. Fig. 23. 25 fig/ml. A good example of the type of mitochondrion found in expiants treated with this hydrocarbon. Notice the parallel cristae (arrow). X 65,000. Fig. 24. 25 fig/ml. A residual (or possibly multivesicular body) surrounded by a unit membrane contains other unit-membraned vesi cles (arrows) and an electron-dense droplet. X 65,000. Fig. 25. 50 /tg/m\. Portion of a cell which shows the cytoplasmic organization near the lumen (L). Note the unit-membraned vacuoles (v) with mucoid material, the scarcity of organized endoplasmic reticulum (er), and the large masses of cytoplasmic filaments (F). An area of the cytoplasm has been enclosed (s) by a circular segment of endoplasmic reticulum (ert). X 73,000. Fig. 26. 25 Mg/ml. Pinocytic vesicles are pictured; their inward or outward direction cannot be ascertained from such photographs. Notice the many free ribosomes (r), the complexity of the cellular connections (ci) and retention of a junctional complex (jc). Diffuse bundles of cytoplasmic filaments (F) course through the cytoplasm. X 41,000. Fig. 27. 50 ng/ml. The complexity of cellular relationships as well as the marked alteration from morphologic states common to res piratory epithelium is evident. Notice the many free ribosomes (r) and the almost structureless mitochondrial profiles (m), x 20,000. Fig. 28. 5 fig/ml. This portion of the cell exhibits a residual body (rb) with stacks of myelin-like lamellae (my) and cytoplasmic seg regation (s). X 47,000.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1968 American Association for Cancer Research. Ultrastructural Alterations Produced by Polycyclic Aromatic Hydrocarbons on Rat Tracheal Epithelium in Organ Culture

E. Roter Dirksen and T. Timothy Crocker

Cancer Res 1968;28:906-923.

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