sample="quota" bates="ZN17256" isource="ctr" decade="1970" class="ui" date="19751005" RESPIRATORY EPITHELIAL LESIONS PRODUCED BY BENZO(A)PYRENE OR AN AIR POLLUTION EXTRACT IN TRACHEOBRONCHIAL MUCOSA FROM PERINATAL HAMSTERS, DOGS AND MONKEYS AND FROM ADULT HUMAN SUBJECTS IN ORGAN CULTURE. T. Timothy Crocker, M.D., Thomas V. O'Donnell, M.D. and Lora L. Nunes, A.B. Polluted air contains a variety of chemical carcinogens , the most commonly recognized of which is benzo(a)pyrene ( ). Benzene-soluble materials from filters used to collect air-borne particles, and fractions of these extracts, not always of known composition, are carcinogenic and may have biologic activity which is relevant to human respiratory carcinogenesis associated with air pollution. In order to learn whether the respiratory mucosa of man (or other primates) can be expected to be more or less susceptible than that of rodents or canines upon exposure of respiratory mucosa of several species to suspected materials under uniform conditions. Tracheobronchial structures of suckling rats and hamsters and of fetal dogs and monkeys have been maintained for 2 to 3 weeks in organ culture. Late fetal or suckling (also referred to as "perinatal") animals were chosen in order to minimize environmental exposure to inhaled air and to make possible the use of whole respiratory structures (Trachea, bronchi) which were small enough to be nourished by diffusion in the organ culture system. In addition, the rapid growth rate of cells of immature tissue was expected to permit more cell generations to occur during the limited time of maintenance of organ cultures. Thus, if more cell divisions could occur after initiation of a potential neoplasm, recognition of a neoplastic transformation might be more likely. The choice of these three orders of mammals was intended to include two orders (rodents and canines) in which experimental bronchogenic carcinoma has been produced by polycyclic environmental chemicals and one order (primates) in which BaP and AP have not bee tested as a cause of bronchogenic carcinoma. Polycyclic hydrocarbons known to be carcinogenic by various methods of testing in rodents have been added to organ culture media and have produced abnormal histological states of respiratory epithelia of sucking rats (3,4,5,6). Among these are: (i) excessive height and crowding of columnar cells to form differentiated columnar cells by one or more layers of undifferentiated pleomophic cells or by stratified cells of a squamous epithelial type; and (iv) loss of all or most epithelial cells. Weakly carcinogenic and non=carcinogenic hydrocarbons have not produced these abnormal sates (6). Whole benzene-soluble extracts of solids from filters used to collect air pollutants (AP) have been applied to this system, and one or more of the abnormal states described above have been induced. Effects of benzo(a)pyrene (BaP) and of AP are described in evaluating the biologic responsiveness of perinatal hamster, dog and monkey respiratory epithelia to a pure compound or an abstract of material under consideration as having possible effects on human health. Presented by T. Timothy Crocker, M.D. at the AMA's Air Pollution Medical Research Conference on October 5, 1970, in New Orleans. Doctor Crocker is Professor of Medicine, University of California, School of Medicine and Research Physician, Cancer Research Institute. In addition to tests with perinatal animals as a source of tracheobronchial structures for culture, adult human subjects undergoing bronchial biopsy or lung resection have yielded some samples for testing. Control culture data for human mucosa are presented more fully elsewhere . Materials and Methods Addition of the whole benzene-soluble air pollution extract (AP) to culture A pool of crude whole benzene-soluble extracts of particulate material from air filters was provided through the courtesy of Dr. John Ludwig, Chief, Laboratory of Engineering and Physical Sciences, National Center for Air Pollution Control, Cincinatti, Ohio. The sample, designated "Miscellaneous Composite Vial #11964" was received as a brown glass vial of tarry material and was stored at 4ºC. This material and the fractions obtained from it were described by Tabor and Smith (8). Samples of AP were transferred to tared glass vials, weighed and exposed to a volume of dimethylsulfoxide (DMSO, Crown-Zellerbach Corporation) or acetone such that there was 180 mgm per ml of solvent. The vials were stoppered tightly and incubated at 37°C overnight. A clear supernate overlay a finely divided suspension which in turn was in contact with coarser particulate material. The entire sample was mixed well and 0.1 ml was transferred with a tuberculin syringe and 27 gauge needle to (i) 6.15 ml of chicken serum, with rapid mixing, and (ii) a pre-weighed glass vial. The serum preparation was stored at 4°C throughout an experiment and when added to medium it represented 25% of the final volume of medium. Results. Respiratory structures exposed to whole benzene-soluble air pollution extract (AP) and benzo(a)pyrene (BaP). Suckling hamster trachea: control cultures. Generally good maintenance of epithelia was observed for as long as 17 days on liquid or clotted acetone control media. (Figures 1-4) but occasionally a zone of undifferentiated epithelium or squamous metaplasia was noted and some devitalized explants occurred (Table 1). Data from both types of media are pooled in the review. Comparison of acetone and DMSO in control liquid media revealed that DMSO favored retention of a columnar cell population but not change in labeling as compared to acetone. While this suggests that DMSO had an effect slightly favoring differentiation, the results from use of both vehicles are pooled in this report. Suckling hamster trachea: treated cultures. AP and BaP reduced the frequency of columnar differentiation and increased the frequency of basal hyperplasia (State 2, Table 1 and Figures 5 and 6) and of pleomorphic undifferentiated states (State 3, Table 1 and see, for example, Figures 7, 8). BaP and AP reduced the level of replication in columnar epithelium except when basal hyperplasia was present (Table 1). AP stimulated DNA synthesis in areas of basal hyperplasia more than BaP did. AP was toxic to hamster tracheas as indicated by increase in the percent of devitalized explants (Table 1). Squamous metaplaisa occurred rarely with BaP or AP (State 4, Table 1 and Figures 9, 10) and was observed in one zone (1%) or 131 states recorded in 124 control explants. Fetal dog tissue at the time of explanting. Respiratory epithelium in fetal dogs was not well differentiated. The columnar cells of trachea had rare cilia and PAS-positive but colloidal iron-negative goblets. Smaller airways were lined by columnar cells lacking mucous inclusions and cilia but filled with finely granulated glycogen. Tritiated thymidine complexes with glycogen and labeling produced a pattern of autoradiographic grains over the cytoplasm generally or over the Golgi zone. Fetal dog tissue: control cultures. Differentiation of columnar epithelium cells occurred during 8 to 17 days in control cultures. Ciliated cells and colloidal iron-positive mucous inclusions in goblet cells appeared in fetal tracheal epithelia, but the fetal dog bronchi did not acquire containing mucous inclusions indicating limits to the extent to which differentiation would be achieved in vitro. Coincident with evolution of differentiated states, the proportion of epithelial cells in DNA synthesis declined. The cells labeled with 3HTdR at the time of planting fell in fetal dog trachea from 3.5 to 1.0%, and in fetal dog bronchi from 5 to 0.6%. Fetal dog tissue: treated cultures. There was a distinct tendency for emergency of basal hyperplasia and pleomorphic epithelial states in control cultures of fetal dog bronchi (Table 1). BaP did not reduce the overall proportion of differentiated epithelial states (States 1 and 2, Table 1) but did increase the frequency of basal hyperplasia with an associated increase in the proportions of labeled nuclei. The effect of AP was different from that of BaP; AP reduced the frequency of differentiated epithelial states, and increased the frequency of squamous metaplasia and pleomorphic epithelia associated with a moderate level of DNA synthesis (Table 1, States 3, 4). Figures 9 and 10 illustrate squamous metaplasia but were photographed from hamster tracheal sections. Fetal monkey tissue at the time of explanting. The state of differentiation of tracheobronchial epithelium was not quite complete in these fetal simians. Not all surface cells bore cilia or mucous inclusions containing acid mucopolysaccharides and were not always arranged in a mature columnar state (Figure 11). The ration of basal to differentiated cells was about 1 to 1 rather than the ration 1 to 2 noted in adults. Fetal monkey tissue: control cultures. Examination at intervals of 8 and 15 days of cultivation revealed progressive differentiation of columnar epithelia which were higher than in planting controls. Pseudostratification occurred and basal cells increased in number, and the proportion of basal to columnar cells became similar to adult ratios in the monkey. Parts of some explants failed to acquire differentiated columnar epithelia and zones of cellular undifferentiated epithelia were present (State 3, Table 1). Cartilage, where present, was well maintained, as were smooth muscle cells and connective tissue elements from which fibroblasts grew into the rayon mesh (Figure 12). Toxicity for hamster trachea, reduction of the proportion of differentiated states in dog tissues and increased proportion of epithelial cells in a higher replicative category without toxicity in monkey tissues are the major effects of this material, demonstrating that BaP, present in AP, does not account alone for biologic effects of AP. This review demonstrates that tracheo-bronchial epithelia from perinatal animals of these 3 orders were altered by BaP and AP to a degree not attributable to cultivation alone, but that all 3 orders did not respond identically to control or test conditions of maintenance in organ culture. The complexity of AP components is too great a to permit a discussion of mechanisms of action but the characteristics and metabolism of BaP are better known and allow at least preliminary discussion of the different responses of the 3 animal orders. One trend in the response to BaP correlates with maturity of the animals whose tissues were explanted. The most mature were human, monkey and hamster and in these there was suppression of DNA synthesis or devitalization. Toxicity of BaP depends on intracellular enzymatic conversion to biologically active derivatives. The aryl hydroxylases responsible for this conversion are present in free-living animals and can be induced only at a fairly advanced state of embryonic development or postnatally. The microsomal enzyme system of which complex. Enzyme activity may be induced to varying degrees by many natural and artificial substrates and enzyme action requires a number of co-factors. The products of metabolism and any one substrate, as BaP, may vary from tissue to tissue and the biologic activity of only a few products (as epoxides, monohydroxy derivatives) has been proven or postulated. Some of the biologic effects of BaP, as toxicity and carcinogenicity, may depend on the degree and specificity of the BaP-hydroxylating activity which can be induced in this group of microsomal enzymes, on the presence of cofactors or inhibitors and on the enzymatic products formed. The greater toxicity of BaP for monkey than for dog tracheo-bronchial epithelia may therefore reflect a genetic difference between orders of mammals or the acquisition by the more nature animal of more readily inducible microsomal enzyme systems which may, in turn, produce BaP metabolites which are toxic in organ culture. The data from adult human respiratory mucosal explants may contribute to either of the latter interpretations, namely, that the toxicity noted in fetal primate epithelium is even more evident in the adult (human) primate or that primates share a response to BaP which is distinct from the response of canines. Significance of Pathologic States of Respiratory Epithelia. Epithelia composed of 3 or more layers of pleomorphic cells appeared occasionally in association with increased labeling. Pleomorphic cells are predominant in dysplastic, presumptively preneoplsatic lesions of the human bronchus. The presumptive human preneoplastic lesions have high proportions of labeled cells both in the fresh state (Figures 17, 18) and after cultivation (Figures 19, 20). Thus, when high labeling is present in a cellular pleomorphic lesion of an animal respiratory epithelium in organ culture, the effect is interpreted as morphologic transformation producing abnormal cells with the potential for autonomous growth. This state could be regenerative, as in repair of injury in vivo, but this is not likely in organ culture where injury usually leaves a single layer of cells. For this reason such an epithelial state in an organ culture is regarded here as potentially preneoplastic; the agent producing it is regarded as potentially carcinogenic. When pleomorphism plus low labeling is produced in treated organ cultures, this state is admitted to represent morphologic alteration of epithelial cells but since it is associated with suppression of DNA synthesis this state is regarded as evidence of toxicity. The appearance and organization of cells in epithelium may not be adequate to define the neoplastic potential of a lesion unless other indices, as replication, are available as well. In all attempts to ascribe neoplastic potential to non-invasive lesions, the validity of the interpretation depends ultimately on showing that similar lesions invade. This finding has not yet been made for lesions produced in organ culture and inferences as to the significance of epithelial lesions are understood to be subject to this ultimate test. The effects of BaP and AP were examined in this report under the assumption that they would produce lesions compatible with the carcinogenic effects in animals, however, hence their effects on respiratory tissue in organ culture are compatible with their ranges of biologic activity in vivo. LEGENDS FOR FIGURES Figures 1-10 are photomicrographs of sucking hamster trachea, Figures 11 - 14 are photomicrographs of fetal M. Cynomalgus and Figures 15 - 20 are photomicrographs of adult human respiratory mucosa. Figures 1, 5, 7, 9 and 11 - 16 are of section stained with colloidal iron- nematoxylin acid mucopolymerase in cartilage matrix and in goblet cells appear grey to black. Figures 2 - 4, 6, 8, 10 are autoradiographs that were stained by the Periodic Acid - Schiff method, dipped in NTB2 photographic emulsion and after development. Figures 17-20 are autoradiographs which were dipped in NTB2 photographic emulsion and stained after development with hematoxylin-cosin. These photomicrographs illustrate the various histologic states referred to in Tables 1 and 2. Suckling hamster tracheas. Figure 1. Trachea at time of explanting. The pseudostratified columnar epithelium (State 1, Table 1, and State 1a, Table 2) is well differentiated with cilia and goblet cells. Note intense mucopolysaccharide staining in cartilage and cellularity of connective tissue. x400. Figure 2. As for Figure 1. The labeling rate in this epithelium is 4.5% x1000. Figure 3. Acetone control, 11 day culture, liquid medium. This columnar epithelium (State 2, Table 1, and State 1a, Table 2) is well differentiated. Its appearance is much the same as it is at the time of explanting. x400 . Figure 4. As for Figure 3. The labeling rate of the epithelium has declined with time in culture and is now 2.9% x1000. Figure 5. Air pollution extract 11 day culture, liquid medium. This highly cellular pseudostratified columnar epithelium exhibits basal cell hyperplasia (State 2, Table 1, and State 1c, Table 2). There is a reduction of cartilage cellularity and mucopolysaccharide staining intensity. x400. Figure 6. As for Figure 5. The irregularity of basal cell size and shape, and the increase in number is more clearly seen. The labeling rate is 2.6% in this epithelium. x1000. Figure 7. Air pollution extract 15 day culture, liquid medium. In this undifferentiated metaplastic epithelium the cells are irregular in size, shape, orientation and staining intensity. This epithelial state is termed pleomorphic undifferentiated (State 3, Table 1, and State 3a, Table 2). There is also some vacuolization of cytoplasm and irregularity in nucleolar staining. x400. Figure 8. As for Figure 7. The pleomorphism of the cells is more apparent. The labeling rate is 2.2% in this epithelium. x1000. Figure 9. Air pollution extract, 8 day culture, clotted medium. In this sqaumous metaplastic epithelium (State 4, Table 1, and State 2b, Table 2) the basal cells are vertically oriented, with flattening of the cells occurring as the luminal surface is approached. There is irregularity of cell size, shape, and orientation, and an indication of intracellular bridges. x400. Figure 10. As for Figure 9. The orientation of cells is more apparent, and variation in staining intensity of nuclei is seen. The labeling rate is 7.0% x1000. Fetal Monkey Trachea. Figure 11. Trachea at the time of explanting. The columnar epithelium is not well differentiated, perhaps because of the age of the fetus, but note intense mucopolysaccharide staining in cartilage and cellularity of connective tissues. x400. Fetal Monkey Bronchi, Clotted Medium Figure 12. Dimethylsulfoxice (DMSO) control, 8 day culture. This pseudostratified columnar epithelium has ciliate as well as goblet cell differentiation. x400. Figure 13. Benzo(a)pyrene (BaP), 15 ug/ml, in acetone, 8 day culture. In this undifferentiated epithelium the cells are irregular in size, shape, orientation and staining intensity. In the lower right corner is a portion of cartilage in which there is loss of cellularity as well as mucopolysaccharde staining X400. Figure 14. Air pollution extract in DMSO, 8 day culture. This undifferentiated epithelium is similar to that produced by BaP (Figure 13). Note the destruction of connective tissue. X400. Adult human bronchial mucosa. Figure 15. Normal respiratory mucosa at the time of explanting. This columnar epithelium (State 1, Table 1, and State 1a, Table 2) has a high proportion of goblet cells in which mucous inclusions appear black. The labeling rate is 0.6% and labeled cells are present only in the basal cell layer. x400. Figure 16. Normal respiratory mucosa cultured for 15 days on control clot medium. This columnar epithelium demonstrates preservation of the state of differentiation observed in Figure 15. The labeling rate was reduced to 0.0%. X400. Figures 17 and 18. Abnormal respiratory mucosa at time of explanting. In the right portion of Figure 13 (x170), differentiated columnar epithelium is present with a labeling rate of 1.6%. In the left portion and in Figure 18 (x400) a dysplastic epithelium, possibly carcinoma in situ (State 2a, Table 2), has labeling rate of 17.1% with labeled cells present at all levels in the epithelium. Figures 19 and 20. Abnormal respiratory mucosa cultured for 11 days on clot medium with BaP or AP. In Figure 19 columnar epithelium with basal cell hyperplasia on the left (labeling rate 10.5%) joins a cellular dysplastic zone regarded as possible carcinoma in situ (State 2a, Table 2) in the right half of the figure. X160. In Figure 20, pleomorphic cells are seen to make up this dysplastic lesion, and DNA synthesis is occurring in 26.6% of cells at all levels in the epithelium. X400.