Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi.

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.

The respiratory epithelium. II. Hamster trachea, bronchus, and bronchioles.

The normal female hamster respiratory epithelium at five airway levels was characterized with the use of coordinated morphologic and histochemical techniques. Five morphologic cell types were recognized in the trachea, stem bronchi, and primary bronchl: basal cells and neurosecretory cells that were basally located and did not reach the lumen and mucous cells [mucous goblet cells and small mucous granule cells (SMGC)], indifferent cells showing mucous-ciliary differentiation, and ciliated cells that reached the lumen. Two epithelial cell types were observed in the bronchioles, ciliated cells and nonciliated Clara cells, both of which reached the lumen. Mucous cells presented as either SMGC with a few small periodic acid-Schiff-positive granules (diastase-resistant neutral mucosubstances) or as goblet cells, filled with the same material. Mucous cells were columnar, and the cytoplasm was electron-dense and contained a well-developed endoplasmic reticulum and Golgi complex. The microvilli of the mucous cells were coated more thickly with colloidal iron than either the cilia or microvilli of ciliated cells. Approximately one-half the cells in the trachea, bronchi, and bronchioles were ciliated. Ciliated cells containing intracellular ciliated cysts with normal cilia projecting into a closed space or ciliated cells bearing compound cilia were observed infrequently. Neurosecretory cells were rarely observed. These cells contained characteristic dense-core granules.

The respiratory epithelium. VI. Histogenesis of lung tumors induced by benzo[a]pyrene-ferric oxide in the hamster.

Lung tumors were induced in female Syrian golden hamsters by intratracheal instillation of benzo[a]pyrene-Fe2O3. The tumors were characterized with the use of coordinated morphologic and histochemical techniques including electron microscopy. The lung carcinomas were classified according to their presumed cell of origin. Most were derived from mucous cells and/or basal cells, and they were classified as either epidermoid carcinomas or as combined epidermoid and adenocarcinomas. The tumors in the second group (57% of the total number of carcinomas) presented a wide spectrum of epidermoid and adeno components. The epidermoid component was characterized in well-differentiated tumors by the presence of intercellular bridges and/or keratinization. Well-developed desmosomes and numerous bundles of tonofilaments were observed ultrastructurally. In diagnosing adenocarcinoma, one no longer needs to depend on the presence of tubules or gross glandular structures as criteria for diagnosis. The presence of intracellular and/or extracellular alveoli, well-developed Golgi complex, and endoplasmic reticulum and/or evidence of mucous secretion provide more definitive criteria. A tumor composed of neurosecretory cells that morphologically resembled a bronchial carcinoid of man was observed. Nests of uniform, small, polygonal cells with round-to-oval nuclei were seen at the light microscopic level. Dense-core secretory granules 1,100-2,200 A were present in the cytoplasm of the tumor cells. Several fibrosarcomas were observed. The tumors showed a very cellular structure, composed of either densely packed ovoid or spindle-shaped cells. Ultrastructurally, the cells resembled fibroblasts. The results obtained in this study give strong support for a histogenetic classification, i.e., a classification based on the cell of origin.

The respiratory epithelium. VII. Epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury.

Regeneration was studied in female Syrian golden hamster tracheal epithelium. The epithelium was focally removed in vivo by scraping it with a blunt probe. At 2 hours, virtually all cells had sloughed from the injured area leaving a bare basal lamina. At 6 and 12 hours, flattened cells that migrated from adjacent uninjured epithelium partially covered the denuded basal lamina. Increased cell division did not occur at these times. Many of the simple squamous cells contained well-developed endoplasmic reticulum, Golgi apparatus, and mucous granules. Other cells resembled basal cells. At 24 hours the defect was covered by one or two layers of simple squamous cells. At that time, many of those cells were in division, and cell division was also greatly increased in mucous cells and basal cells in the uninjured epithelium distant from the defect. At 48 hours the epithelium was stratified, composed of four or five layers of polygonal to flattened cells, typical of nonkeratinizing epidermoid metaplasia. The cells contained many tonofilament bundles, a large Golgi apparatus, and many tiny mucous granules. Mitoses were seen in all cell layers. At 72 hours, the surface layer of cells was undifferentiated (indifferent cells) overlying an epithelium that otherwise retained its epidermoid character. Indifferent cells were characterized by an electron-lucent cytoplasm and a lack of tonofilament bundles, mucous granules, or cilla. Cells similar in other respects to indifferent cells were seen that possessed mucous granules or early signs of cilla formation. Some cells showed mucous granules and cilla developing in the same cell. By 96 hours, the regenerated epithelium was fully differentiated and was indistinguishable from the normal epithelium. These observations show that mucous cells have a significant role in the regenerative response. Mucous cells have a dual potential; they can undergo epidermoid metaplasia and still retain the ability to secrete mucus. The study explains the universal occurrence of mucosubstances in areas of epidermoid metaplasia and makes more understandable the previously reported fact that many bronchogenic carcinomas are combined epidermoid and adenocarcinomas. In the presence of a carcinogen, the hypothesis has been forwarded that initiation of mucous cells and basal cells occurs, which leads to malignant transformation and produces tumors that show active secretory activity and keratinization, often in the same cell.

The respiratory epithelium. I. Human bronchus.

Six morphologic cell types comprise the human bronchial epithelium: basal cells that do not reach the bronchial lumen, neurosecretory cells (Kulchitsky's cells, K-cells, or small granule cells) that rarely reach the lumen, and indifferent cells, mucous cells [small mucous granule cells (SMGC) and mucous goblet cells], ciliated cells, and ciliated-mucous cells that do reach the lumen. Ciliated-mucous cells bearing fully developed cilia and containing mucous granules are seen only occasionally. Three of the cell types that reach the lumen are microvillus covered and do not bear cilia. The microvillus-covered nonciliated cells are: 1) neurosecretory cells, 2) indifferent cells, and 3) mucous cells. Neurosecretory cells contain characteristic dense core granules. Such cells rarely reach the lumen. Indifferent cells are rarely seen. They have a pale cytoplasm and show no evidence of either ciliary or mucous differentiation. Similar cells are observed showing early signs of either ciliary or mucous differentiation or even both types of differentiation in the same cell. Mucous cells comprise the vast majority of microvillus-covered cells. They present either as SMGC with a few small mucous granules or as goblet cells, filled with mucus. These columnar cells are characterized ultrastructurally by dense cytoplasm and a well-developed endoplasmic reticulum and Golgi apparatus. The microvilli are coated with a glycocalyx that binds colloidal iron more avidly than that of either cilia or microvilli of ciliated cells. Possible interrelationships between the different cell types in normal epithelium are discussed.

Metabolism of acyclic and cyclic N-nitrosamines in cultured human bronchi.

The metabolism of carcinogenic N-nitrosamines was studied in normal-appearing bronchial specimens obtained from 4 patients. Explants of bronchi were cultured in a chemically defined medium for 7 days. N-Nitrosamines [N-nitrosodimethylamine (DMN), N-nitrosodiethylamine (DEN), N,N'-dinitrosopiperazine (DNP), N-nitrosopyrrolidine (NPy), and N-nitrosopiperidine (NPd)] labeled with 14C were each then added at 100 mumoles for 24 hours. Measurable CO2 was formed by bronchial explants from: 1) DMN, DEN, and NPy in all 4 patients; 2) DNP in 3 of 4 patients; and 3) NPd in only 1 of 4 patients. In all bronchial specimens, these N-nitrosamines and/or their metabolites bound to bronchial mucosal DNA and protein. Binding levels were higher to protein than to DNA. Binding levels of DNP were as high as those with the two acyclic N-nitrosamines DMN and DEN, but binding levels of NPy and NPd were lower. Human bronchus was shown to metabolize and bind acyclic and cyclic N-nitrosamines found in the environment and in tobacco smoke.

The respiratory epithelium. III. Histogenesis of epidermoid metaplasia and carcinoma in situ in the human.

The histogenesis of epidermoid metaplasia and carcinoma in situ was analyzed in human bronchial epithelium. The conclusion is that epidermoid metaplasia and carcinoma in situ can result from conversion of mucous cells. This implies the direct transformation of one type of fully differentiated cell to another. The study therefore emphasizes the differentiation potentialities of the mucous cells that can divide and undergo goblet cell hyperplasia and epidermoid metaplasia. Epidermoid metaplasia is a common reaction to injury in the bronchus. In our series of cases it was especially frequent in patients without neoplastic disease who had undergone intratracheal intubation or tracheostomy and who had been maintained on a respirator in the Shock Trauma Unit, University of Maryland. Future studies will be required to distinguish the difference, if any, between epidermoid metaplasia destined to become malignant carcinoma and that which is not. One difference noted in this study was the absence of overt cornification in epidermoid metaplasia in patients without neoplastic disease.

The respiratory epithelium. V. Histogenesis of lung carcinomas in the human.

One hundred human primary lung carcinomas were studied by light and electron microscopy and by light microscopic histochemistry to demonstrate mucosubstances. The tumors were classified histogenetically and were grouped into three major categories depending on their cell of origin: 1) tumors from basal and/or mucous cells; 2) tumors from neurosecretory cells; and 3) tumors from Clara cells. Most carcinomas (88%) arose from basal and/or mucous cells. These were subdivided into epidermoid carcinomas (21%), combined epidermoid and adenocarcinomas (46%), and adenocarcinomas (21%). The criteria for epidermoid differentiation included the presence of tonofilament bundles, poorly developed endoplasmic reticulum and Golgi apparatus, and well-developed desmosomes. The criteria for adeno differentiation included well-developed endoplasmic reticulum and Golgi apparatus, poorly developed desmosomes, the presence of extracellular and/or intracellular alveoli, and/or other evidence of cellular secretion such as secretory granules. In adenocarcinomas with extracellular alveoli, typical junctional complexes were also present at the luminal aspect where the cell apexes bordered the alveolus. With these criteria, combined epidermoid and adenocarcinomas were the most common type of lung carcinoma. We anticipate that the new data will clarify categories such as small cell anaplastic carcinoma and large cell carcinoma of the World Health Organization classification. In addition, the histogenetic classification of lung tumors may be of value in the future in studies of risk factors, prognosis, prevention, and treatment of lung cancer.

Long-term survival of normal adult human tissues as xenografts in congenitally athymic nude mice.

Bronchi, pancreatic ducts, and colons from adult human were maintained as xenografts in congenitally athymic nude N:NIH(S) mice for 715, 145, and 89 days, respectively. After an ischemic crisis and revascularization of the human tissue, the epithelium regenerated to a normal differentiated morphology. The long-term survival of normal adult human epithelial tissues as xenografts provides model systems for the study of the interactions of chemical and/or physical carcinogens with human tissues.

Long-term organ culture of human uterine endocervix.

Explants of human endocervix have been maintained in organ culture for 24 weeks. The epithelium was viable for the entire duration of culture, as demonstrated by ultrastructural morphology and mitotic activity. The epithelium of the explants retained a near normal morphology for 2 to 4 weeks. The only changes observed were decreased mucus secretion in columnar cells, focal epidermoid metaplasia, and an increase in autophagic vacuoles. Subsequently, a slowly progressive transformation of the columnar epithelium into a metaplastic epithelium was observed, with loss of mucus-secreting columnar cells and ciliated cells and the appearance of cuboidal and flattened epidermoid cells, forming often two to three layers. Metaplasia began at the top of the papillae and ridges and extended progressively downwards into the clefts. Nevertheless, in a few areas deep in the clefts, columnar cells retained evidence of mucus secretion during the entire duration of culture. Epidermoid metaplasia appeared to develop in the explants by transformation of columnar mucous cells into cuboidal and flattened cells with epidermoid characteristics. This hypothesis is supported by ultrastructural observations that showed mucus secretion and early keratinization in the metaplastic cells. Mitotic figures were observed with transmission electron microscopy for up to 24 weeks, and all dividing cells contained mucous granules.

Morphological and cytokinetic responses of hamster airways to intralaryngeal or intratracheal cannulation with instillation of saline or ferric oxide particles in saline.

The morphological and proliferative effects of intratracheal cannulation (ITC) or intralaryngeal cannulation (ILC), with or without the instillation of saline or Fe2O3 particles in saline, were studied in Syrian golden hamsters. Instillation of Fe2O3 in saline at either airway level resulted in a similar distribution of Fe2O3 particles in all lung lobes. ILC produced laryngeal mucosal wounds. ITC produced laryngeal and tracheal mucosal wounds. The cannula-induced wounds were associated with proliferative epithelial lesions. ITC, but not ILC, resulted in significant increases in the mitotic rates (MR, 6-h colchicine blockade) of tracheal epithelial cells at 24 and 32 h postcannulation. Instillation of saline by ITC produced slight increases in intrapulmonary bronchial and bronchiolar MR, but saline given by ILC did not increase MR at any airway level. Instillation of Fe2O3 particles in saline by ITC produced increases in tracheal, intrapulmonary bronchial, and bronchiolar MR. Instillation of Fe2O3 particles in saline by ILC had little effect on tracheal MR, but increased intrapulmonary bronchial and bronchiolar MR. Foci of Fe2O3 particle-laden macrophages were associated with mild bronchiolar-alveolar hyperplasia at the junctions of the terminal bronchioles and the alveolar ducts. The cytokinetic and morphological changes in the intrapulmonary airways were associated with the influx of inflammatory cells in response to Fe2O3 particle deposition. The marked increases in tracheal MR and the localized hyperplastic tracheal epithelial lesions were clearly associated with mechanical wounding from the cannula during ITC. Comparative studies using ILC or ITC instillation techniques allowed further investigations of the important role of tracheal mucosal wounding in the induction of respiratory carcinogenesis, as described in a companion paper (Keenan et al., Cancer Res., 49: 1528-1540, 1989).

Multifactorial hamster respiratory carcinogenesis with interdependent effects of cannula-induced mucosal wounding, saline, ferric oxide, benzo[a]pyrene and N-methyl-N-nitrosourea.

The carcinogenic response induced in the respiratory tract of Syrian golden hamsters by repeated intratracheal instillations of benzo[a]pyrene (BP) adsorbed to ferric oxide (Fe2O3) particles suspended in saline, is shown to result from the interactions of these factors and cannula-induced tracheal wounding. Previous acute studies of intratracheal cannulation (ITC) versus intralaryngeal cannulation (ILC) showed that tracheal cell proliferation increased significantly in ITC-induced mucosal wounds. Only mild increases in intrapulmonary cell proliferation were produced by Fe2O3-saline given by ILC or ITC (Keenan et al., Cancer Res., 49: 1521-1527, 1989). The present chronic studies included the following variables: a single instillation by ILC of N-methyl-N-nitrosourea (MNU) at 5 weeks of age; 15 weekly treatments (beginning at 7 weeks of age) by ILC or ITC alone, or with instillations of saline, or Fe2O3-saline, or BP-Fe2O3-saline; and appropriate controls. Repeated ITC-induced tracheal wounds caused persistent tracheal epithelial hyperplasia, metaplasia and/or atrophy and submucosal fibroplasia during the observation period of 22 to 78 weeks of age (the time of terminal sacrifice). Tracheal cancers (in situ or invasive carcinomas) were seen only in those hamsters which had received repeated ITC and one or both carcinogens. The cancer latency was shortest and the incidence of tracheal (50%) and main-stem bronchial (21%) cancers highest in hamsters given MNU and repeated ITC with BP-Fe2O3-saline. Hamsters given carcinogens by ILC (which induced laryngeal but not tracheal wounds) developed proliferative lesions and cancers of the larynx but no tracheobronchial cancers. These data show the singular importance of repeated ITC-induced intratracheal wounding as an enhancing factor in this respiratory carcinogenesis model. The findings suggest that the mechanism of tumor enhancement involves not only changes in target epithelial cell proliferation, but also alterations in normal epithelial-mesenchymal interactions during tracheal regeneration from repeated chronic submucosal inflammation and mesenchymal repair. In the present experimental model, a single dose of MNU at 5 weeks of age, repeated instilled doses of BP, and tracheal mucosal wounding were each found to be important determinants of the carcinogenic response. Additional effects were observed for instilled Fe2O3 particles, and possibly saline. Interplay of all these factors, as well as of genetic, nutritional, and infectious factors, are considered in relation to risk assessment and prevention.

Long-term organ culture of human bronchial epithelium.

Human bronchial epithelium has been maintained in organ culture in serum-supplemented medium for 4 months. After 4 to 6 weeks in culture, various changes in morphology were apparent. There was an increase in autophagic vacuoles in mucous, ciliated, and basal cells, a reduction in the height of the columnar cells, a decrease in the number of goblet mucous cells, and an increase in cells with small mucous granules. After 3 months in culture, the basal lamina was frequently covered by 2 or 3 layers of epithelial cells consisting of nonkeratinizing squamous cells with short microvilli and small mucous granules. Less frequently, keratinizing squamous cells were seen. Differentiated epithelium incorporated precursors into macromolecules in serum-free medium, supplemented with vitamin A, at 1 week of culture. These explants exhibited changed epithelium by 2 weeks, similar to that described for epithelium in serum-supplemented medium after 4 to 6 weeks.

Metabolism of dimethylnitrosamine and 1,2-dimethylhydrazine in cultured human bronchi.

The metabolic activation of several chemical classes of procarcinogens is being studied in cultured human bronchi. Previous studies have shown that carcinogenic polynuclear aromatic hydrocarbons are metabolically activated by the bronchial epithelium. In the study reported here, dimethylnitrosamine and 1,2-dimethylhydrazine were also found to bind to both cellular DNA and protein. Bronchial DNA was methylated in both the O-6 and N-7 positions of guanine. In addition to polynuclear aromatic hydrocarbons, an aliphatic nitrosamine and a methylhydrazine can now be added to the list of xenobiotic chemical carcinogens metabolized by human bronchus.

Binding of (3H)benzo(a)pyrene to DNA in cultured human bronchus.

The studies reported here demonstrate some of the factors affecting the binding of benzo(a)pyrene (BP) to macromolecules in cultured human bronchial mucosa. Bronchial specimens were obtained at either surgy or "immediate" autopsy from patients with and without lung cancer. Grossly normal-appearing pieces of bronchus were cultured in a chemically defined medium, i.e., CMRL 1066 medium containing 1 mug insulin per ml, 0.1 mug beta-retinyl acetate per ml,, 0.1 mug hydrocortisone hemisuccinate per ml, 2 mM L-glutamine, 100 units penicillin G per ml, and 100 mug streptomycin per ml. After 7 days, explant cultures were exposed to [3H]BP, usually for 24 hr, and then binding to total cellular macromolecules was studied by autoradiography, and binding to DNA was measured following isolation of DNA from bronchial mucosal cells. The extent of binding of [3H]BP was dependent on dose of BP, length of exposure to [3H]BP, and temperature. By autoradiography, bronchial epithelial cells bound more [3H]BP than stromal fibroblasts. Both 7,8-benzoflavone and butylated hydroxytoluene appeared to reduce the level of [3H]BP bound to DNA, while nicotine apparently did not alter the level of binding. These studies demonstrate that the bronchial mucosa, an important human cancer target tissue, has the capability to form metabolites of BP which bind to macromolecules including DNA. In addition, 7,8-benzoflavone and butylated hydroxytoluene, both known to alter the microsomal metabolism of BP, reduce the level of [3H]BP bound to DNA.

Optical properties of normal and injured cells. Application of cytographic analysis to cell viability and volume studies.

A cell spectrophotometer (Cytograf Model 6300 A, Bio/Physics Systems, Inc.) was tested in a cytotoxic assay using Ehrlich ascites tumor cells as a model system. Several cellular conditions associated with volume expansion, staining of cellular components and fixation of cells were applied and the magnitude of the scattering and extinction signals were tested in these diverse cellular conditions. The magnitude of the scattering pulse of a cell spectrophotometer was found to be greatly dependent on the staining color and intensity of cellular components with vital dyes or following osmium tetroxide fixation. When the absorption wavelength of the vital dye was close to the wavelength used in the cell spectrophotometer (about 630 nm), dead stained and living nonstained cell populations were completely separated from each other. The magnitude of the extinction pulse was greatly dependent on the state (normal, injured cells) and staining intensity (vital dye staining, osmium fixation) of cellular components. The magnitude of the extinction pulse was reduced from that in normal cells when cells were treated with p-chloromercuribenzene sulfonic acid or in a hypotonic solution that caused a marked volume expansion of injured cells. When cells were fixed with a mixture containing glutaraldehyde and osmium tetroxide the cellular components of normal and injured cells turned black and in these conditions the magnitude of the extinction signal was in a linear correlation to the cross-sectional area of cells. In the present study, the cell spectrophotometer proved to be an efficient method for estimation of cellular viability, based on different scattering properties of cells, offering the advantages of high speed and precision. Demonstration of the use of a variety of vital dyes having diverse extinction properties with capabilities to differentiate between living and dead cells has indicated the potential use of the cell spectrophotometer in cytotoxic assay. Modification of the magnitude of the extinction signal in the cell spectrophotometer also shows great promise for accurate automated size determination of both normal and injured cells. Previously, determination of the size of injured cells has been beset with methodologic errors.

Effects of fixation and postfixation treatments on volume of injured cells;.

The effects of fixation with glutaraldehyde (GA), formaldehyde (FA), glutaraldehyde-formaldehyde (GA-FA), flutaraldehyde-osmium tetroxide (GA-Os04) and osmium tetroxide (OsO4) on cel volume were studied in control, p-chloromercuribenzene sulfonic acid (PCMBS)-treated and hypotonically-treated Ehrlich ascites tumor cells. Among the variables investigated were concentration of the tixative agent, osmolity of the buffer, total osmolaity of the fixation solution, osmolaltity of the postfixation buffer and the time of fixation and postfixation treatment; in addition, the effects of adding calcium and high molecular weight compounds to the fixative solution were studied. When the effects of standard fixatives on control, PCMBS- and hypotonically-treated cells were compared, marked differences were apparent in the behavior of control and injured cells. Control cells retained near prefixation volume in 3% GA and 3% GA-1% OsO4, swelled in 4% FA or 1% OsO4 and phosphate buffer (tkrp), whereas tpcmbs (310 mosM KRP)- and hypotonically-treated cells (103 mos M KRP) shrank in all aldehyde fixatives but swelled in 1% OsO4. Reducing the buffer osmolality had similar effects on control and injured cells although, there were variations in degree...

Modulation of glycogen stores in epithelial cells during airway development in Syrian golden hamsters: a histochemical study comparing concanavalin A binding with the periodic acid-Schiff reaction.

We studied glycogen storage in the developing airway epithelium of Syrian golden hamsters from gestational Day 11 to neonatal Day 2 using concanavalin A (ConA) staining as an adjunct approach to the periodic acid-Schiff (PAS) reaction. One hundred and fourteen fetuses and neonates were fixed in 4% formaldehyde-1% glutaraldehyde, 6% mercuric chloride-1% sodium acetate-0.1% glutaraldehyde, and 95% ethanol, embedded in paraffin, and stained with ConA-horseradish peroxidase conjugate as well as with PAS. ConA staining was abolished by alpha-glucosidase digestion or by pre-treatment with periodic acid, demonstrating that ConA bound to glycogen. In tissues fixed with mercury and/or aldehydes, ConA staining was greatly enhanced by pepsin digestion. Airway glycogen stores, revealed by ConA and PAS, fluctuated during development. At first all the undifferentiated epithelial cells contained abundant glycogen. Then, coincident with the appearance of the first endocrine cells, the glycogen stores were depleted. Thereafter, glycogen accumulated in pre-secretory and basal cells until birth, but by 2 days after birth the glycogen stores were again depleted. The initial depletion of glycogen followed by repletion was observed at all levels of the conducting airways; changes in the trachea preceded those in the bronchi and bronchioles by 1 and 2 days, respectively.

Chemical carcinogenesis in the tracheobronchial epithelium.

Some of the recent work in pulmonary carcinogenesis is briefly reviewed. Morphologic studies of neoplastic and preneoplastic lesions of the human bronchi are compared with similar studies of carcinogenesis and epithelial regeneration in the hamster trachea. These studies suggest that bronchogenic carcinomas are typically complex mixtures of three basic phenotypes, the epidermoid and the mucous and dense-core granulated (endocrine) phenotypes. Pure forms of these phenotypes are rare, as different cells and even individual cells in single tumors express more than one phenotype. The clinical significance of such phenotypic variability is not yet known. Bronchial cell types which retain the capacity to divide include the mucous cell, the basal cell and perhaps the dense-core granulated cell. Studies of epithelial regeneration and preneoplastic lesions suggest that the mucous cell may be pivotal both in the response to injury and in carcinogenesis. Cigarette smoking is believed to be the major etiologic factor in bronchogenic carcinoma. Cigarette smoke contains initiators of carcinogenesis, but it contains a plethora of probable promoters and cocarcinogens as well. It is hypothesized that cigarette smoke may both initiate bronchial cells and promote carcinogenesis in cells which have previously been initiated by smoke or other factors. It is further hypothesized that that mucous cell is the major target for initiation and subsequent tumorigenesis. The ultimate phenotype(s) displayed by the tumor is suggested to result from the effect of microenvironmental factors upon the initiated cell and its progeny.

An unusual bronchial carcinoid tumor: light and electron microscopy.

An unusual bronchial carcinoid tumor was studied by light and electron microscopy. The tumor cells, which appeared to be monotonously uniform in hematoxylin and eosin stained sections, were found to be morphologically heterogeneous at the ultrastructural level with regard to the size, number, and morphology of the endocrine granules. Presumptive endocrine granules were seen in all tumor cells, but some cells contained only small round granules (2000 A largest diameter), other cells contained large round granules (some with as large a diameter as 1.0 mu), and some cells contained large polymorphic granules. Many of the cells stained positively at the light microscopic level when selective stains for endocrine cells were applied. All types of granules showed argyrophilia at the ultrastructural level. Numerous clusters of endocrine cells were observed in the otherwise normal bronchial and bronchial glandular epithelium. The spectrum of granule morphologies, as seen in the tumor cells, was displayed in cells of the intraepithelial clusters. Some mucous cells and sparsely ciliated cells within these clusters contained argyrophilic granules. Multiple continuities existed between the epithelial endocrine cell clusters and the underlying tumor mass. The intraepithelial clusters represent foci of carcinoma in situ, the genesis of which is discussed.