Influence of growth factors and medium composition on benzo[a]pyrene- and vitamin A-induced cell proliferation and differentiation in hamster tracheal epithelium in organ culture.

Tracheal organ cultures and isolated tracheal epithelial cells are frequently used to study effects of carcinogens and retinoids on both proliferation and differentiation of respiratory tract epithelial cells. For each of these in vitro models, optimal culture conditions have been established, varying in type of culture medium and composition of growth factor and hormone supplementation, which by themselves may influence cellular proliferation and differentiation. In this study, we investigated the influence of medium composition and growth factor supplementation on the effect of benzo[a]pyrene (B[a]P) and vitamin A on cellular proliferation and differentiation in hamster tracheal epithelium in organ culture. In tracheae cultured in Ham's F12 medium, cell proliferation was decreased by B[a]P relative to untreated controls, whereas vitamin A in combination with B[a]P increased cell proliferation compared with that in tracheae treated with B[a]P alone. The effects in tracheae cultured in CMRL-1066 medium were just the opposite: B[a]P increased cell proliferation and vitamin A decreased B[a]P-induced proliferation. To explain this difference in cell proliferation, the effects of various growth factors (epidermal growth factor and transferrin) and medium components (nucleotides, NAD(+)/NADP and CaCl(2).2H(2)O) on B[a]P and vitamin A-induced cell proliferation were investigated. The main factor responsible for the different effects on cell proliferation appeared to be the concentration of Ca(2+) in the culture medium; addition of CaCl(2).2H(2)O to Ham's F12 medium resulted in effects of B[a]P and vitamin A on cell proliferation comparable with those observed in tracheae cultured in CMRL-1066 medium. These results clearly show that the composition of the culture medium, and particularly the concentration of Ca(2+), strongly influences the effect of vitamin A and B[a]P on cell proliferation in hamster tracheal epithelium in organ culture.

Epidermal cell proliferation and terminal differentiation in skin organ culture after topical exposure to sodium dodecyl sulphate.

Epidermal cell proliferation and differentiation were investigated in vitro after exposure to the anionic surfactant sodium dodecyl sulfate (SDS). Human skin organ cultures were exposed topically to various concentrations of SDS for 22 h, after which the irritant was removed. Cell proliferation was measured immunohistochemically by incorporation of bromodeoxyuridine (BrdU) into the DNA of cells during S-phase, while the expression of transglutaminase and involucrin were used as markers of differentiation. Cell proliferation was moderately increased at concentrations of SDS that did not affect the histomorphology (0.1% and 0.2% SDS). A marked increase of cell proliferation was observed 22 to 44 h after removal of SDS at a concentration (0.4%) that induced slight cellular damage. Exposure of human skin organ cultures to a toxic concentration of SDS 91.0% led to decreased cell proliferation. Transglutaminase and involucrin were expressed in the more basal layers of the epidermis after exposure to 0.4% or 1.0% SDS. Moreover, intra-epidermal sweat gland ducts were positive for transglutaminase at these irritant concentrations. These in vitro data demonstrate that SDS-induced alterations of epidermal cell kinetics, as described in vivo are at least partly due to local mechanisms and do not require the influx of infiltrate cells. However, we were unable to relate to altered cell kinetics to the release of interleukin-1 alpha or interleukin-6. Furthermore, supplementation of the culture medium with 12-hydroxyeicosantetraenoic acid did not affect epidermal cell proliferation. Rabbit skin cultures appeared more sensitive to SDS than human skin. At nontoxic doses, the irritant induced an increase of epidermal cell proliferation, similar to that observed in human skin discs.

Expression of insulin-like growth factor II in spontaneously immortalized rat mesothelial and spontaneous mesothelioma cells: a potential autocrine role of insulin-like growth factor II.

Insulin-like growth factors (IGFs) are polypeptides that play an important role in cellular proliferation and differentiation. The present study examines the role of IGFs in the growth of mesothelial cells. Cell lines derived from normal rat mesothelium as well as lines derived from spontaneous rat mesotheliomas were found to express RNA transcripts for IGF-II. In contrast, cell lines derived from asbestos-induced rat mesotheliomas did not express this growth factor. All cell lines expressed receptors for IGF-I and IGF-II, as well as insulin receptors. Coexpression of IGF-II and its cognate receptor suggested that IGF-II was acting as an autocrine growth factor in the spontaneously immortalized cells and the cells derived from the spontaneous tumors. The biological activity of IGF-II secreted by the cell lines into conditioned medium could be neutralized using an IGF-II-specific antibody. Growth was inhibited in a dose-dependent manner; at the highest antibody concentration used (100 micrograms anti-IGF-II/ml), cell growth was decreased to 47% of control values. This inhibition was partially reversible by treatment of the cultures with IGF-II (91% of the control). These data suggest that IGF-II expression may be involved in the spontaneous alteration of rat mesothelial cells and may function as an autocrine or paracrine growth factor to modulate the growth of these cells in vitro and in vivo. Ubiquitous expression of IGF-II by cells that have not been exposed to asbestos and the lack of IGF-II expression by asbestos-transformed cells suggest that the mechanisms of changes in growth factor expression differ in mesothelial cells transformed by different pathways.

Relation between benzo[a]pyrene-DNA adducts, cell proliferation and p53 expression in tracheal epithelium of hamsters fed a high beta-carotene diet.

Vitamin A and beta-carotene protect against respiratory tract cancer by inhibiting the formation of DNA damage and controlling cellular proliferation and differentiation. Recently, it has been shown that the p53 tumor-suppressor gene plays a crucial role in the etiology of respiratory tract cancer. In the present study, we investigated the relationship between benzo[a]pyrene (B[a]P)-DNA adducts, cell proliferation and p53 expression and the possible effect of beta-carotene on such a relationship in tracheal epithelium of hamsters given intratracheal instillations of B[a]P-Fe2O3 particles suspended in saline. DNA-adducts were quantified by the 32P-postlabeling assay, cell proliferation was quantified by immunocytochemical detection of incorporated BrdU during S-phase, and p53 protein was detected by immunohistochemistry with an antibody that recognized both the wild-type and the mutated protein (BioGenex, Clone BP53-12-1). A clear relationship appeared to exist between the extent of B[a]P-DNA adduct formation, the induction of cell proliferation and the expression of p53 protein in hamster tracheal epithelium. These results suggest that B[a]P induces cell proliferation in hamster tracheal epithelial cells most likely by the induction of mutations in the p53 gene. Furthermore, beta-carotene was not found to influence the formation of B[a]P-DNA adducts, which is probably due to the high B[a]P dose. Moreover, beta-carotene did not statistically significantly affect cell proliferation and p53-protein expression in hamster tracheal epithelial cells.

Vitamin A and beta-carotene influence the level of benzo[a]pyrene-induced DNA adducts and DNA-repair activities in hamster tracheal epithelium in organ culture.

Although most studies concerning the effect of vitamin A and beta-carotene on chemical carcinogenesis are focused on tumour promotion and progression, these compounds may affect initiation as well. In this study the influence of vitamin A and beta-carotene on unscheduled DNA synthesis (UDS) was investigated in hamster tracheal epithelium in organ culture exposed to benzo[a]pyrene (B[a]P). DNA-repair activities were compared with the level of B[a]P-DNA adducts as measured both by 32P-postlabeling and by immunocytochemical detection. In hamster tracheal epithelial cells, both vitamin A and beta-carotene significantly increased B[a]P-induced UDS, with 40% and 45%, respectively. At the same time, vitamin A and beta-carotene decreased the level of B[a]P-DNA adducts in these cells with 18% and 40%, respectively as measured by 32P-postlabeling and with 12% and 35%, respectively as measured by immunocytochemistry. The effect of vitamin A on B[a]P-induced UDS and DNA-adduct levels in hamster tracheal epithelium appeared to depend on the dose of B[a]P vis-à-vis the concentration of vitamin A. The results of the present study show that both vitamin A and beta-carotene cause a decrease in B[a]P-DNA adduct levels by enhancing DNA-repair activities. Because the formation of B[a]P-DNA adducts is considered to be an early step in respiratory tract carcinogenesis, it is suggested that enhancement of DNA-repair activities by vitamin A and the subsequent removal of DNA adducts may be one of the mechanisms involved in vitamin A-mediated protection against cancer.

A critical appraisal of intratracheal instillation of benzo[a]pyrene to Syrian golden hamsters as a model in respiratory tract carcinogenesis.

Several experimental models have been developed to study respiratory tract carcinogenesis. The most widely applied in vivo model uses Syrian golden hamsters which receive intratracheal instillations of a suspension of benzo[a]pyrene (B[a]P) particles attached to ferric-oxide (Fe2O3) particles in saline; it was first described by Saffiotti et al. [1]. This model has several benefits compared with other experimental models; however, the large number of variables affecting the tumour response is a clear disadvantage because the tumour response is difficult to control. In this review, we describe a systematic analysis of various variables that may influence the tumour response of the respiratory tract with the aim to further standardize the method and increase, through that, its suitability and predictability. The most important variables influencing the tumour response, as shown by statistical analysis of 29 representative studies, turned out to be the administered dose and the particle size. Both these variables influence the actual dose and the contact-time of the B[a]P particles with the target cells. The present study does not support the widespread opinion that ferric-oxide particles enhance the tumour response of the respiratory tract. In conclusion to the present analysis, some recommendations are made which probably increase the predictability of the model.

Benzo[a]pyrene-induced respiratory tract cancer in hamsters fed a diet rich in beta-carotene. A histomorphological study.

The effect of a high dietary level of beta-carotene on the formation of preneoplastic and neoplastic respiratory tract lesions was studied in hamsters intratracheally treated with benzo[a]pyrene (B[a]P) attached to ferric oxide (Fe2O3) and suspended in saline. In addition to conventional histopathological examinations, the expression of cytokeratins and the glutathione S-transferase isoenzyme Pi (GST-Pi) was determined in tracheal epithelium using immunocytochemical techniques. B[a]P treatment increased the expression of cytokeratins in tracheal mucous and ciliated epithelial cells as detected by antibody RCK102 (cytokeratins 5 and 8), which normally recognizes basal cells only. The expression of cytokeratins in mucous and ciliated cells as detected by antibody RGE53 (cytokeratin 18) was decreased by B[a]P treatment. Furthermore, the expression of the cytokeratin detected by antibody RKSE60 (cytokeratin 10), characteristic of metaplastic squamous cells, and the expression of the GST-Pi, characteristic of metaplastic changes, was increased in trachael epithelium of hamsters treated with B[a]P. There was no evidence for dietary beta-carotene affecting the expression of cytokeratins or GST-Pi. The incidence of preneoplastic changes and tumors of the respiratory tract was not reduced by dietary beta-carotene. On the contrary, the tumor response of the respiratory epithelium was almost twice as high in hamsters fed the high beta-carotene diet than in hamsters on the low beta-carotene diet. However, this difference was not statistically significant (P = 0.15); hence, the present study did not produce evidence for a clear effect of beta-carotene on B[a]P-induced respiratory tract cancer in hamsters.

Mesothelial cell proliferation induced by intrapleural instillation of man-made fibers in rats and hamsters.

Long-term inhalation exposure to a biopersistent man-made ceramic fiber (RCF 1) results in a high incidence of pleural mesotheliomas in Syrian golden hamsters but not in identically exposed rats. To understand better the mechanisms involved in the intraspecies pathobiology of fiber-exposed mesothelium, the ability of the two different man-made fibers to induce cell proliferation in hamster and rat pleural mesothelial cells was investigated. Three dose levels of either glass fibers (MMVF 10) or ceramic fibers (RCF 1) were instilled intrapleurally into male Fischer 344 rats and male Syrian Golden hamsters. Rats and hamsters were exposed to approximately equal numbers of long thin fibers per kilogram of body weight using a single intrapleural instillation. Bromodeoxyuridine (BrdU) was administered via an implanted osmotic pump, and mesothelial cell proliferation was assessed at 7 and 28 days postinstillation (PI) using immunocytochemical visualization of labeled S-phase cells. Both rats and hamsters exhibited dose-dependent increases in proliferation of pleural mesothelial cells following exposure to both fiber types. Interspecies differences in mesothelial cell proliferation were noted for fiber type and pleural site. At 28 days PI, RCF-induced mesothelial cell proliferation was found to be more pronounced in hamsters than in rats in the caudal visceral pleural. Comparing both fibers either by equal mass or by equal fiber numbers, mesothelial cell proliferation in RCF 1-treated animals was higher than in animals exposed to MMVF 10, especially in hamsters, and may be a factor in the difference in mesothelioma induced by the two fibers. The higher sustained (28 day) mesothelial cell proliferation in the visceral pleural of hamsters exposed to RCF may contribute to the species-specific differences in mesothelioma incidence found in long-term rodent inhalation studies.

Pleural lesions in Syrian golden hamsters and Fischer-344 rats following intrapleural instillation of man-made ceramic or glass fibers.

The mesothelium is a target of the toxic and carcinogenic effects of certain natural mineral and man-made fibers. Long-term inhalation of a ceramic fiber (RCF-1) results in a high incidence of pleural mesotheliomas in Syrian golden hamsters but not in identically exposed Fischer-344 rats. The present study compared the histopathology of the early pleural response in rats and hamsters instilled with artificial fibers. Groups of Syrian golden hamsters and Fischer-344 rats were instilled with ceramic (RCF-1) or glass (MMVF-10) fibers directly into the pleural space. Each species received approximately equal numbers of long, thin fibers per g body weight. Fiber-induced lesions were compared 7 and 28 days postinstillation. Both hamsters and rats developed qualitatively similar dose-dependent inflammatory lesions that were not fiber-type specific. Both species developed fibrosis in conjunction with inflammation in the visceral pleura, but a striking interspecies difference was noted in the pattern of mesothelial cell response. Hamsters developed greater surface mesothelial cell proliferation and had focal aggregates of mesothelial cells embedded deep within regions of visceral pleural fibrosis. It is hypothesized from the present study that the marked fiber-induced proliferative mesothelial cell response of the hamster visceral pleura may explain the high number of pleural mesotheliomas found in long-term fiber studies in this species.

DNA adduct formation and repair in hamster and rat tracheas exposed to benzo[a]pyrene in organ culture.

Syrian golden hamsters are much more susceptible than Wistar rats to the induction of tracheal tumors by benzo[a]pyrene (B[a]P). To investigate whether this difference is reflected in the pattern of DNA adduct induction and removal, tracheas from either species were isolated and exposed to B[a]P (5 micrograms/ml) in organ culture. At various time-points B[a]P-DNA adducts were quantified by 32P-postlabeling; unscheduled DNA synthesis (UDS) and cell proliferation were determined by [3H]thymidine incorporation during the 18 h before sampling. In an induction-repair experiment tracheas were exposed to B[a]P for 2 days, and cultured for another 4 days without B[a]P. After 2 days of exposure total B[a]P-DNA adduct levels were 10 times higher in hamster compared to rat tracheas. In hamster tracheas one major adduct was formed (95%), namely the adduct between (+)-anti-benzo[a]pyrene diolepoxide and deoxyguanosine (BPDE-N2dG). In rat tracheas BPDE-N2dG comprised approximately 60% of the total B[a]P-DNA adduct level. The other major adduct found in rat tracheas is probably derived from interaction of syn-BPDE and deoxyadenosine. During exposure to B[a]P in hamsters the adduct level increased to 36 +/- 19 adducts/10(6) nucleotides (add/10(6)n) on day 2. Two days after removal of B[a]P the B[a]P-DNA adduct level had decreased to 60% of that on day 2; there was no further decrease in the B[a]P-DNA adduct level, despite considerable cell proliferation at the end of the 6 day culture period. UDS increased during exposure to B[a]P and decreased after removal of B[a]P. In rats removal of B[a]P did not lead to a decrease in the B[a]P-DNA adduct level, which agreed with the observed absence of UDS. In a second experiment tracheas were exposed to B[a]P continuously for 15 days. In hamster tracheas the total B[a]P-DNA adduct level increased from 11 +/- 0.7 add/10(6)n after 1 day of exposure to 105 +/- 2 add/10(6)n after 15 days; also UDS increased with increasing exposure until day 11. Cell proliferation was low at the end of the culture period. In rat tracheas no progressive increase in the B[a]P-DNA adduct level was seen, UDS was not increased and cell proliferation had increased significantly at the end of the exposure period. The extent of adduct induction in the trachea of the two species corresponded with the different susceptibilities to B[a]P-induced tumor formation.

High survival rate of hamsters given intratracheal instillations of benzo[a]pyrene and ferric oxide and kept on a high beta-carotene diet.

The study described in this paper was primarily conducted to identify the cell types involved in the formation, progression and regression of metaplastic changes in the respiratory tract epithelium of hamsters after intratracheal intubations with benzo[a]pyrene. Furthermore, the role of vitamin A and beta-carotene in these processes was studied. In the course of the study a remarkable effect of dietary beta-carotene on survival of hamsters became a subject of investigation. Hamsters were fed diets with various levels of vitamin A or beta-carotene and were treated intratracheally with a suspension of benzo[a]pyrene with ferric oxide in saline. The tumour response of the respiratory tract was very low (2.8%) and hyper- and metaplasia of respiratory epithelium were virtually absent. However, an interesting observation was an exceptionally low mortality of only 2% after 69 weeks in the group of hamsters fed a high beta-carotene diet (1% w/w), whereas in the other groups mortality after 69 weeks amounted to 25%. Although the exact cause of death of most of the hamsters could not be established, a 40% reduction of lipid peroxidation in the livers was found in the high beta-carotene group. Moreover, in this group the degree and incidence of nephrosis and of focal mineralization of kidneys and heart were lower than in the other groups. These favourable effects of the high beta-carotene diet may have contributed to the unusually high survival rate in hamsters fed this diet. Further studies are planned to verify and study this observation.

Expression and modulation of adhesion molecules on human bronchial epithelial cells.

Epithelial damage in the airways is a feature often observed in patients with asthma and is probably caused by the interaction of epithelial cells with leukocytes. As adhesion molecules are thought to be important in this interaction, we analyzed the expression and modulation of adhesion molecules on primary cultured human bronchial epithelial cells and the bronchial epithelial cell lines BEAS-2B and NCI-H292. E-selectin, P-selectin, and VCAM-1 were absent under basal and stimulated conditions. The adhesion molecules ICAM-1 (CD54), LFA-3 (CD58), and CD44 (H-CAM) were expressed basally on primary cultured human bronchial epithelial cells and the BEAS-2B and NCI-H292 cell lines. CD44 and LFA-3 expression did not change after stimulation with IFN-gamma or TNF-alpha. In contrast, ICAM-1 expression on human bronchial epithelial cells and BEAS-2B cells could be increased by incubation with PMA, IFN-gamma, TNF-alpha, and especially with the combination of IFN-gamma and TNF-alpha. The maximal ICAM-1 expression on both epithelial cell types was obtained with the combination of TNF-alpha and IFN-gamma after 48 h of incubation. The NCI-H292 cell line was different in that it only showed increased ICAM-1 expression after stimulation with PMA and IFN-gamma and not by the combination of IFN-gamma and TNF-alpha or with TNF-alpha alone. In conclusion, the bronchial epithelial cells tested express several adhesion molecules, but only ICAM-1 expression was influenced by inflammatory cytokines.

Effect of coffee drinking on cell proliferation in rat urinary bladder epithelium.

A possible effect of freshly brewed drip coffee on urinary bladder carcinogenesis was investigated in male Wistar rats using cell proliferation in urinary bladder epithelium as the indicator of tumour promotion. Male rats were given either undiluted coffee brew (100% coffee), coffee diluted 10 times (10% coffee) or tap water (controls), as their only source of drinking fluid for 2 or 6 wk. Uracil, known to induce cell proliferation in urinary bladder epithelium, was included in the study as a positive control. In rats receiving 100% coffee, body weights, liquid intake and urinary volume were decreased. Neither histopathological examination of urinary bladder tissue nor the bromodeoxyuridine labelling index revealed biologically significant differences between rats receiving coffee and the tap water controls. Uracil increased the labelling index and induced hyperplasia of the urinary bladder epithelium, as expected. It was concluded that these results produced no evidence that drinking coffee predisposes to tumour development in the urinary bladder.

Comparative 32P-postlabeling analysis of benzo[a]pyrene--DNA adducts formed in vitro upon activation of benzo[a]pyrene by human, rabbit and rodent liver microsomes.

An interspecies comparison was made of the DNA-adducts formed in vitro upon incubation of rat liver DNA (RL-DNA) with benzo[a]pyrene (BP) in the presence of liver microsomes. Incubations were carried out with RL-DNA, BP (100 microM) and liver microsomes from hamsters, mice, rabbits, rats, 3-methylcholanthrene (3MC) pretreated rats and from humans. To analyse the adduct profiles, the 32P-postlabeling technique with the nuclease P1-enhancement procedure was used. The total amount of adduct formed varied greatly with the species; also the number of adduct spots detected was different, ranging from 1 to 5. In all incubations the BP-N2-deoxyguanosine adduct was formed. Relative to the total adduct level, the level of this adduct varied from 26% with rat, 54% with hamster, 56% with 3MC-pretreated rat, 58% with mouse and 75% with rabbit, to 100% with human liver microsomes. In human liver microsomes both the total amount of cytochrome P-450 per mg microsomal protein and the ethoxyresorufin O-deethylation (EROD) activity were low compared to that in animal liver microsomes. In microsomes from 3MC-pretreated rats the EROD activity was strongly induced. There was no correlation between EROD activity in non-induced microsomes and total adduct level. To compare BP-DNA adduct formation in human white blood cells (WBC) with that in RL-DNA, WBC were incubated with BP and 3MC-pretreated rat microsomes. The adduct profile in WBC-DNA differed from that observed after incubation of RL-DNA: the BP-N2-deoxyguanosine adduct in WBC-DNA accounted for 97% of the total adduct level. It is concluded that the 32P-postlabeling method is a suitable technique to investigate both qualitative and quantitative differences in BP-DNA adduct formation between species. Furthermore, the incubation of microsomes from the liver (or other sources) with a genotoxic agent and isolated DNA or cells can be a useful approach to study the formation and stability of reactive intermediates that are able to bind to DNA, also with respect to differences between species or tissue.

Formation and repair of benzo[a]pyrene-DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis.

Hamster tracheal organ cultures were used to investigate the relationship between DNA adduct formation measured directly by the 32P-postlabeling assay, and the DNA damage measured indirectly by the unscheduled DNA synthesis (UDS) assay. Hamster tracheas were treated with three concentrations of benzo[a]pyrene (B[a]P) for 2 days. Postlabeling and UDS assays were also carried out a few days after removal of the B[a]P. Furthermore, the types of B[a]P-DNA adducts formed in the in vitro organ culture were qualitatively compared with those formed in vivo after intratracheal intubation of B[a]P attached to Fe2O3 particles. In vivo only one adduct was detected by 32P-postlabeling. This adduct cochromatographed with the trans-addition produce of dG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). In vitro, a clear B[a]P-DNA adduct pattern was also found with the 32P-postlabeling assay. Four different adducts were found. The main adduct spot migrated to the same position on the thin-layer chromatogram as the in vivo adduct. B[a]P-DNA adduct formation was both time- and dose-dependent. During the first day after removal of B[a]P the adduct levels still increased, thereafter they decreased at all B[a]P concentrations. A time- and dose-dependent increase in UDS was observed in the tracheal epithelial cells treated with B[a]P in vitro. After removal of the B[a]P, UDS decreased immediately, in contrast to the formation of DNA adducts. The results of the present study show that B[a]P induces time- and dose-dependently both DNA adducts and UDS in hamster tracheal organ culture. Moreover, the main DNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same as that found in vivo.

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide.

The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-3H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 micron, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-3H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration.

Effect of retinol and cigarette-smoke condensate on dye-coupled intercellular communication between hamster tracheal epithelial cells.

The dye-coupled intercellular communication across gap junctions in primary hamster tracheal epithelial cells has been studied in serum-free, hormone-supplemented medium. In the absence of vitamin A, non-cytotoxic concentrations of cigarette-smoke condensate (CSC) inhibited intercellular communication between tracheal epithelial cells in a concentration-dependent way. All-trans retinol and retinoic acid showed biphasic effects on intercellular communication depending on their concentration. Physiological concentrations of retinol and retinoic acid increased the dye-coupled transfer of Lucifer Yellow CH via gap junctions compared with the dimethylsulfoxide-treated tracheal epithelial cells. At pharmacological concentrations retinol slightly increased the intercellular communication in the first 2 h of the exposure period, whereas upon longer treatment times with retinol and retinoic acid, gap-junction-mediated intercellular communication was inhibited almost completely. When retinol was given to tracheal epithelial cells before exposure to CSC or simultaneously with CSC-exposure, retinol counteracted the inhibitory potential of CSC on intercellular communication. The results of the present study clearly indicate that both CSC and all-trans retinol influence the intercellular communication between primary hamster tracheal epithelial cells in serum-free, hormone-supplemented culture medium.

Intermediate filament expression in normal and vitamin A depleted cultured hamster tracheal epithelium as detected by monoclonal antibodies. A study with emphasis on histological changes.

Using immunohistochemical techniques, the keratin expression patterns in basal and columnar cells (mucus-producing and ciliated cells) were investigated in tracheal organ cultures. Tracheas were from either hamsters fed a control diet or from hamsters fed a vitamin A-deficient diet; tracheas from the latter group were treated in vitro with all-trans retinol. In tracheas from hamsters fed a control diet, basal cells generally reacted with the RCK102 antibody and columnar cells with the RGE53 and the HCK19 antibodies, and both basal and columnar cells were recognized by the RCK105 antibody. The squamous cell cytokeratin 10 (detected by the RKSE60 antibody) was not expressed in cultured tracheas from hamsters fed a normal or a vitamin A-deficient diet. In the course of the in vitro period a number of keratins were "switched on" or "switched off" in both basal and columnar cells. In tracheas from vitamin A-deprived hamsters the RCK102 antibody clearly recognized basal cells and cigarette smoke condensate-induced proliferating basal cells, whereas the RGE53 antibody reacted with mucus-producing and ciliated cells. During organ culture foci of columnar epithelial cells expressed basal cell properties (detected with the RCK102 antibody) after all-trans retinol treatment and were found negative for the RGE53 antibody. Furthermore, it appeared that the RGE53-negative columnar cells contained periodic acid-Schiff-positive mucous granules. These findings indicate that basal cells may differentiate into columnar cells. Tracheal epithelium did not appear to co-express vimentin next to keratins during organ culture, which may be due to the intact three-dimensional organization present in these organ cultures.

Effect of cigarette smoke condensate and vitamin A depletion on keratin expression patterns in cultured hamster tracheal epithelium. An immunohistomorphological study using monoclonal antibodies to keratins.

Keratin expression in hamster tracheal epithelium was investigated during organ culture in serum-free, hormone-supplemented medium using monospecific monoclonal antibodies. Generally, tracheal basal cells expressed keratins detected by antibodies RCK102 and RCK103, while columnar epithelial cells were stained positively by RGE53, RCK103, RCK105 and HCK19. Metaplastic squamous cell foci reacted with antibodies RKSE60, RCK103 and HCK19. Early metaplastic alterations were more clearly RKSE60-positive than the mature lesions. In the vitamin A-depleted tracheas basal cells were clearly RCK102-positive. Superficial cells in the central part of areas of squamous metaplasia induced by cigarette smoke condensate expressed the basal cell keratins, and were negative for the columnar cell keratin 18 detected by the RGE53 antibody. This finding suggests that in cigarette smoke condensate-induced squamous metaplasia basal cells play an important role. The mucus-producing cells at the edges of metaplastic squamous cell foci expressed the keratins specific to columnar cells. Cigarette smoke condensate exposure accelerated epithelial keratinization compared to the vitamin A-depleted epithelium. It was concluded that not only small mucous granule cells, but also basal cells are involved in the development and maintenance of induced squamous metaplasia in tracheal epithelium. Furthermore, in vitro vitamin A-depleted epithelium did not coexpress vimentin in addition to the different keratins.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. I. A cell proliferation study.

The effects of cigarette smoke condensate (CSC) and all-trans retinol on the cell proliferative activity of vitamin A-deprived hamster tracheal epithelium have been studied in vitamin A-deficient, serum-free, hormone-supplemented medium in organ culture. In the absence of retinol, CSC induced a dose-dependent increase in labeling index (LI) during 12 days of culture. The basal cells were more sensitive to CSC exposure than non-basal cells during the first 6 to 8 culture days. However, in squamous metaplastic foci developing after culture day 6, both basal and non-basal cells in the mid-part of the epithelium were labeled. Physiological concentrations of all-trans retinol stimulated the non-basal LI and inhibited the basal cell LI. Compared with dimethylsulfoxide (DMSO), all retinol concentrations used in the present study inhibited the basal cell LI at each time point examined (4-12 days culture). Exposure of tracheal rings to retinol, either before or after exposure to CSC, or simultaneous exposure to retinol and CSC, clearly decreased the CSC-induced basal cell proliferative activity depending on the retinol concentration used. It is concluded from the present study that squamous metaplasia induced by vitamin A-deficiency or by CSC originates mainly from basal cells and that for the maintenance of these lesions, both basal and non-basal cells play a role. Furthermore, all-trans retinol inhibited CSC-induced basal cell proliferation.