Ha-ras oncogene mutations in cell lines derived from rat tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene.

The frequency of Ha-ras mutations was determined as a function of neoplastic progression in cell lines derived from rat tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene. Restriction fragment-length polymorphism (RFLP) analysis revealed an A----T transversion in the second base of codon 61 in 2 of 11 cell lines. One of the positive cell lines was tumorigenic, but the other was neither tumorigenic nor anchorage independent, thus indicating a lack of correlation between neoplastic stage and ras mutation. Densitometry analysis of the RFLP bands indicated that approximately 50% of the cells within these two heterogeneous populations contained the mutation. Direct sequence analysis of polymerase chain reaction-amplified DNA confirmed these results and did not reveal any other mutations in this region of the Ha-ras gene.

[3H]benzo[a]pyrene utilization in rats following tracheal implant exposure.

Open-ended rat tracheal implants (OETI) were exposed to 40 micrograms [3H]benzo[a]pyrene (B[a]P)-gelatin pellets and the 3H activity in the OETI, the host's tissues and excretia was determined 3-96 h after insertion of the pellets. The radioactivity in the OETI reached near peak activity by 3 h, and decreased almost 10-fold by 24 h. Most of the activity was associated with parent B[a]P throughout the 95 h. The 3H activity in the surrounding tissue also was mostly associated with B[a]P, but the 3H activity in the liver, kidney, blood and urine was mostly associated with water-soluble plus conjugated metabolites. In the feces, 68% of the 3H activity was in B[a]P at 3 h, but mostly organic as well as water-soluble plus conjugated metabolites were extracted from it throughout the remaining 96 h. Forty-eight hours after insertion of the B[a]P pellets, the feces contained almost 16% of the total 3H activity. Pre-exposure of the OETI to B[a]P for 4 days before insertion of the [3H]B[a]P pellets stimulated metabolism of B[a]P in the tracheas approximately 2-fold, but had no significant effect on the host tissues.

Oncogene expression in cell lines derived from rat tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene.

Expression of four oncogenes and two keratin genes was determined in rat tracheal epithelial cell lines derived from tracheal implants exposed in vivo to 7,12-dimethylbenz[a]anthracene. Cell lines were grouped into four stages of neoplastic progression based on phenotypic markers in order to correlate oncogene expression with stage of malignancy. Northern analysis of RNA revealed a significantly enhanced expression of the c-myc oncogene in the most tumorigenic or tumor-derived cell lines, whereas preneoplastic cells expressed approximately five-fold less transcript. Southern analysis of tracheal cell DNA did not demonstrate amplification of the c-myc gene in any of the positive cell lines. In contrast to c-myc, other oncogenes such as ras and fos were expressed in all cell lines, as well as in control cell cultures, to a similar extent. Patterns of differentiation were examined in these epithelial cell lines by determining the expression of two distinct keratin genes, KA-1 and KB-2. Both malignant and preneoplastic cells expressed the KB-2 gene at variably high levels, whereas the expression of the KA-1 keratin was barely detectable in any of the cell lines. The stage-specific expression of the c-myc oncogene in these tracheal cell lines suggests a correlation between the regulation of certain oncogenes and neoplastic progression in this model of respiratory carcinogenesis.

Benzo[a]pyrene- and formaldehyde-induced DNA damage and repair in rat tracheal epithelial cells.

The single and combined effects of 2 environmental carcinogens, benzo[a]pyrene (BAP) and formaldehyde (HCHO), on cell growth and DNA damage were assessed in a rat tracheal epithelial cell line, C18. Treatment of C18 cells with HCHO for 90 min reduced the calculated growth index at the highest concentration tested, 400 microM, while no growth effects were observed with BAP treatments. Combination treatments reduced the growth index to 75% of control values. Alkaline elution analysis of C18 cell DNA detected both DNA-protein crosslinks (DPC) and single-strand breaks (SSB) as a result of HCHO treatment, while BAP caused only SSB. HCHO-induced SSB were repaired within 2 h, whereas BAP-induced SSB persisted for at least 48 h. Combination treatment of cells with BAP followed by HCHO enhanced the number of SSB, but reduced DPC. The results are discussed in reference to earlier work which demonstrated the interaction in vivo between BAP and HCHO with respect to their carcinogenicity in rat tracheal implants.

Growth inhibition and DNA damage induced by benzo[a]pyrene and formaldehyde in primary cultures of rat tracheal epithelial cells.

The effects of benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 microM BAP for 24 h or 200 microM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by cornified envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.

The detection of DNA-protein cross-links in rat tracheal implants exposed in vivo to benzo[a]pyrene and formaldehyde.

The DNA damage associated with benzo(a)pyrene (B[a]P) and formaldehyde (HCHO) exposure in rat tracheal implants was determined by alkaline filter elution adapted to measure DNA-protein cross-links (DPC) in vivo. In addition, histopathological responses of the tracheal epithelium were quantitated after multiple exposures to 20 micrograms B[a]P and 0.2% HCHO. Compared to either agent alone, combined exposure for 1-4 weeks caused an increase in cellular atypia and greater thickness of hyperplastic and metaplastic lesions. HCHO exposure resulted in a dose-dependent increase in DPC with a maximal response of 85% DNA filter retention at 0.2% HCHO, which were mostly removed by 72 h. B[a]P did not cause DPC, but when tracheas were pre-exposed to 20 micrograms B[a]P followed by 0.05% HCHO there was a 15% decrease in HCHO-induced DPC. This competition between B[a]P and HCHO for sites presumably on DNA does not offer a clear explanation for their markedly enhanced cocarcinogenicity observed in previous studies, but does demonstrate the interaction between the two agents in tracheal epithelium.

The induction of growth-altered cell populations (tumor-initiation sites) in rat tracheal implants exposed to benzo[a]pyrene and formaldehyde.

The effects of exposure to benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on the induction of carcinogenesis in rat tracheal implants was determined as the number of growth-altered cell populations (tumor-initiation sites, TIS) per trachea. While exposure twice-weekly for 4.5 months to 0.2% HCHO solution gave only a weak response (0.25 TIS/trachea), 2.37 TIS per trachea were detected after exposure to 20 micrograms BAP in the same regimen. The combination of BAP followed by HCHO had a greater response than either agent alone (7.83 TIS/trachea), while the reverse exposure gave 1.49 TIS per trachea, which was less than BAP alone. Thus, the induction of TIS by combined exposure to BAP and HCHO was dependent on the order of exposure, and could not be predicted from their individual exposures.

Quantitation of growth-altered cell populations (tumor-initiation sites) induced in open-ended rat tracheal implants by split doses of benzo[a]pyrene.

The numbers of tumor-initiation sites (TIS) induced by split doses of benzo[a]pyrene (BAP) in open-ended rat tracheal implants (OETI) were quantitated as the average number of growth-altered cell populations isolated per carcinogen-exposed OETI. These growth-altered cell populations were identified as primary cell cultures which survive in medium lacking a pyruvate supplement, a condition in which normal tracheal cells die. The OETI were exposed to an average total dose of 1450 micrograms BAP, which was delivered twice weekly from 170-, 76- or 41-micrograms BAP-gelatin pellets over 4.5, 9 or 18 weeks respectively. OETI exposed for 4.5 or 9 weeks had 0.33 TIS/trachea when growth-altered cell populations were selected at 14 days from the first and second outgrowth cultures. An additional 1.0-1.17 TIS/OETI were detected if selection was delayed until 63 days in the third and fourth outgrowth cultures. In contrast, 2.0 TIS/trachea were selected after 14 days of culture from OETI pre-exposed for 18 weeks, and an additional 2.50/trachea were detected if selection was delayed to 28-63 days of culture. The TIS from this last group were also distinguished by more rapid growth and a large number of multiple cell cultures from the same explant, which indicated that more than one TIS was present on the explant and/or the growth-altered cell population had expanded enough to appear in more than one outgrowth culture. These growth-altered cell populations had also acquired a high degree (83%) of subculturability, i.e. immortality. These results suggest that OETI exposed repeatedly for a long duration had many TIS of which many had received multiple hits necessary for rapid progression of neoplasia. A large number of TIS were induced by a 4.5-week exposure, when the total BAP dose was doubled to 2934 micrograms. In this case a total of 5.75 TIS were induced per OETI. However, the cell cultures grew slowly, few multiple cultures were obtained, and only 52% of the growth-altered cell populations were subculturable.

Sequential appearance of anchorage independence, uncontrolled nuclear division and tumorigenicity in 7,12-dimethylbenz(a)anthracene-exposed rat tracheal epithelial cells.

Epithelial cell lines derived from rat tracheal implants 2 and 9 months after a 4-week exposure to 200 micrograms dimethylbenz(a)anthracene-beeswax pellets, and previously assayed for growth in soft agarose and tumorigenicity, were tested at the same time of subculture for cytochalasin B-induced multinucleation to determine the relationships between anchorage-independent growth, uncontrolled nuclear division, and tumorigenicity. The relationships among the three phenotypic markers could be separated into five distinct groups. Group I cell lines showed no growth in agarose, showed no cytochalasin-induced multinucleation, and formed no tumors in nude mice. Group II cell lines exhibited anchorage independence but were negative for the other markers. Group III cell lines were anchorage independent and exhibited a positive response to cytochalasin B (more than 10% of the cells had three or more nuclei), but were tumor negative. Group IV cell lines were positive for all three markers. Group V cell lines grew in soft agarose, were cytochalasin B negative, but formed tumors only 4 months after the cell inoculations. The 20 cell lines generated 2 months after carcinogen exposure distributed in the groups as follows: Group I, 20%; Group 2, 20%; Group III, 50%; Group IV, 15%; and Group 5, 10%. The 27 cell lines generated 9 months after carcinogen exposure distributed among the groups as: Group I, 4%; Group II, 18%; Group III, 26%; Group IV, 52% and Group V, 0%. The results indicate that: anchorage independence precedes the two other markers of growth autonomy; uncontrolled nuclear division appears as a separate property after anchorage independence and before tumorigenicity; tumorigenicity appears preferably in the cell populations that exhibit anchorage independence and uncontrolled nuclear division; and progression in growth autonomy occurs in the tracheal implants in vivo which can be detected in vitro as an increase in cell lines positive for the three phenotypic markers.

Pathological changes induced by formaldehyde in open-ended rat tracheal implants preexposed to benzo(a)pyrene.

The promotion effects of 0.1% formaldehyde (HCHO) in phosphate-buffered saline (PBS) were tested in rat tracheal implants preexposed to a minimal carcinogenic dose of 468 micrograms benzo(a)pyrene (BAP) released over one month from 865 micrograms BAP-beeswax pellets. At the time of pellet removal, the tracheas were made into open-ended, flow-through, tracheal implants (FTTI), and exposed twice/week to HCHO for 30 weeks. Morphological alterations in the FTTI were monitored biweekly by collection of exfoliated cells from the luminal washings for cytopathologic diagnosis, and periodically by sacrificing animals for histopathology. FTTI exposed to the BAP followed by 30 weeks of HCHO had extensive squamous metaplasia, a high proliferation index of 7.87 [3H]thymidine-labeled cells/mm basement membrane, and foci of moderate and marked atypia. Clear diagnosis of some of the lesions was difficult because of the acute toxic effects of the repeated exposures to HCHO. These effects were seen in the tissues as well as in the exfoliated cells, which attest to the latter as an efficient, non-destructive, method for determining the responses of the tracheas to exposure to toxic and carcinogenic agents. FTTI exposed to BAP followed by twice weekly PBS, had a mostly flattened epithelium, and a low proliferation index (0.39). FTTI exposed to beeswax pellets, followed by the HCHO had a relatively high proliferation index (4.20) in a mucociliary epithelium exhibiting some basal cell hyperplasia. Control FTTI had a normal mucociliary epithelium with a proliferation index of 1.52 [3H]thymidine labeled cells/mm basement membrane.

Progression-linked properties of specific-lesion cell populations from 7,12-dimethylbenz[a]anthracene-exposed rat tracheal implants.

The purpose of this study was to determine whether there are any relationships between the morphological expressions of the progression of neoplasia in tracheal epithelium in vivo, and the increased in vitro growth autonomy expressed by carcinogen-altered cell populations isolated from the same tracheas. Rat tracheal implants were exposed for 2 weeks or 4 weeks to 200 micrograms 7,12-dimethylbenz[a]anthracene (DMBA)-beeswax pellets. In the first phase of this study reported earlier (1), the numbers of carcinogen-altered cell populations, identified by their lack of need for exogenous pyruvate and insulin for survival in cell culture, were quantitated 2, 6 and 9 months after the start of the exposures. Before generating the cell cultures, lesions on the pieces of pre-exposed tracheal implants were identified by placing them in organ culture for 24 h and collecting the exfoliated cells from the medium for diagnostic cytopathology. Although the pieces of 2-week DMBA-exposed tracheas had not developed any markedly atypical lesions by 9 months, there was a progressive increase in the growth autonomy of the small number of subculturable cell populations (approximately 1/trachea) obtained from this exposure group. This was seen in the decreased time needed in culture to expand the population for testing anchorage-independent growth and tumorigenicity in nude mice. Also, anchorage-independent growth was markedly enhanced from 25 to 80% between 2 and 6 months and then to 100% at 9 months. Tumorigenicity did not show an increase. In the 4-week DMBA-exposed group, 74% of the large number of subculturable cell populations (approximately 5.0/trachea) already showed anchorage-independent growth at 2 months. This percentage increased to 94-100% at 6 and 9 months. A progressive increase in tumorigenic cell populations was also clearly seen. At 2 months, 26% of the cell populations inoculated into nude mice formed tumors. This number increased from 45 to 61% at 6 and 9 months, respectively. The pieces of tracheas taken at 2 months after the start of the 4-week DMBA exposure had no markedly atypical lesions. At 6 months one marked atypia was detected, and at 9 months five markedly atypical lesions and two carcinomas in situ were present. The cell populations derived from the explants harboring these lesions showed a high incidence of growth autonomy, e.g. anchorage-independent growth and tumor formation, indicating a close relationship between these properties and the evolving morphological manifestations of tumor development.

Utilization of pyruvate and pyruvate precursors by normal and carcinogen-altered rat tracheal epithelial cells in culture.

The metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen-altered cells derived from dimethylbenz(a)anthracene-exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate-supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three- to four-fold higher incorporation of radioactivity from [2-14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U-14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two-fold greater glucose consumption and two-fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two-fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U-14C]glutamine or [U-14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2 was distinctly different between NPC and SPC except for the 1.7-fold higher utilization of [1-14C]glucose along the oxidative arm of the pentose cycle in the normal cells.

Malic enzyme and malate dehydrogenase activities in rat tracheal epithelial cells during the progression of neoplasia.

Malic enzyme and malate dehydrogenase (MDH) activities were radiometrically assayed in digitonin fractionated normal primary cultures (NPC), preneoplastic selected primary cultures (SPC) and tumor-derived primary cultures (TPC) of rat tracheal epithelial cells. Carcinogen-altered SPC and TPC selectively grow in the absence of pyruvate, which is required by NPC for survival. Mitochondrial-containing particulate fractions from TPC and especially SPC had markedly higher levels of NADP+-dependent malic enzyme than NPC in the presence or absence of pyruvate. This suggests that induction of mitochondrial malic enzyme activity occurs early in the progression of neoplasia. Malic enzyme activities in the soluble fractions from the various populations were not distinctly different. In contrast, particulate-bound MDH activity was higher in NPC and SPC than TPC in most cases, indicating a decrease in this enzyme late in tumorigenesis.

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells.

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.

Hexose uptake in 7,12-dimethylbenz(a)anthracene-preexposed rat tracheal epithelial cells during the progression of neoplasia.

Hexose uptake during the progression of neoplasia in rat tracheal epithelial cells was studied by measuring the uptake of 2-deoxy[3H]glucose (2-dGlc) in nontumorigenic (C-18) and tumorigenic (T-8, 1000-WT) rat tracheal epithelial cell lines with varying degrees of cell association as well as in: normal primary cell cultures (NPC) derived from explants of nonexposed tracheas; selected primary cell cultures (SPC) generated from explants of 7,12-dimethylbenz(a)anthracene-treated tracheal implants; and primary tumor cell cultures (TPC) derived from explants of 7,12-dimethylbenz(a)anthracene-induced tracheal carcinomas. The latter two groups represented cells from earlier and late stages in the progression of neoplasia, respectively, and each displayed an in vitro growth advantage that allowed for their survival and growth in medium devoid of supplements of pyruvate and insulin. This property was used in this study to select the carcinogen-altered cells from neighboring normal cells. Uptake of 2-dGlc per microgram of DNA was similar in subconfluent cultures of all cell lines. At confluency, uptake per microgram of DNA was reduced markedly (greater than 3-fold) in C-18 cells but it was reduced only 1.3-fold in T-8 cells and 1.6-fold in 1000-WT cells. Hexose uptake was further reduced in T-8 and 1000-WT cell cultures generated as outgrowths from explants of denuded tracheas bearing a reestablished epithelium from each cell line. Under these conditions, T-8 cells retained higher 2-dGlc uptake than did C-18, but uptake by 1000-WT was lower, indicating that tissue-like cell associations have a profound effect on hexose uptake in these epithelial cells. Results were generally similar when uptake was expressed per mg of protein although, in several instances, the interpretation of uptake data was affected by differences in the protein content between cultures (assessed by comparing protein:DNA ratios). Compared to NPC, hexose uptake was lower in SPC and one group of TPC. A second group of TPC, characterized by loose cell associations and much cell overlapping, had distinctly higher 2-dGlc uptake than did controls. Comparable results in these primary cultures were also observed when the number of cells per culture was used as a reference for 2-dGlc uptake. Under conditions of glucose deprivation, hexose uptake was increased in NPC and SPC. The production of lactic acid in each type of culture was dependent on the level of glucose in the medium, and this was nearly 2-fold greater in NPC than in SPC.(ABSTRACT TRUNCATED AT 400 WORDS)

Selection of carcinogen-altered rat tracheal epithelial cells preexposed to 7,12-dimethylbenz[a]anthracene by their loss of a need for pyruvate to survive in culture.

Normal rat tracheal epithelial cells require exogenous pyruvate to survive in culture while carcinogen-altered cells do not. The aims of our study were, (i) to utilize this loss of requirement for pyruvate by carcinogen-altered cells as a way of selecting out and quantitating the numbers of altered cell populations induced in tracheas exposed to 200 micrograms 7,12-dimethylbenz[a]-anthracene (DMBA)-beeswax pellets for different lengths of time, (ii) to determine the constancy of these numbers at increasing time intervals after the exposure, (iii) to look for distinctive growth properties in cell populations that may be related to the exposure conditions. Tracheal implants were exposed for 2 weeks, 4 weeks or continuously to the 200 micrograms DMBA. At 2, 6 and 9 months after the start of exposure, the tracheas were cut into pieces and primary cultures established from outgrowths in an enriched Waymouth's medium. After 2 weeks, pyruvate and insulin were removed from the medium for 2 weeks to kill the normal cells and leave the altered, i.e., the selected cell populations (SPC). The pyruvate and insulin were then returned to the medium to stimulate rapid expansion of the SPC. The length of DMBA exposure had a marked effect on the number of SPC induced in the tracheas. There were 1.8 SPC/trachea 2 months after the 2 weeks exposure, while 5.0 and 7.2 SPC were obtained from the 4 weeks and continuously exposed tracheas, respectively, at this time. The numbers of SPC did not increase with time after exposure indicating that the maximal number of initiation sites was fixed. Between 2 and 9 months the number of subculturable SPC increased from 67 to 88% in the 2 week exposed group, while 93-100% of the SPC were subculturable after the longer DMBA exposures. Only the SPC from the 2 week DMBA--2 month group were distinguished by smaller numbers of cells surviving the selection medium and by a slower average growth rate compared to all the other experimental conditions.

Heterotopic tracheal transplants: techniques and applications.

Heterotopic tracheal transplants, placed subcutaneously in syngeneic rats have been extensively used in our laboratory. The objective of these experiments was to study the toxic and/or carcinogenic effect of several compounds on the respiratory tract mucosa. This was attained by exposing the transplants to an intraluminal pettet containing the toxicant or carcinogen mixed with an adequate matrix (gelatin, beeswax, stearyl alcohol, silastic, etc.). By varying the concentration of the test chemicals, it is possible to study dose-response relationships, and by changing the pellet matrix, the effects of release rate (dose rate) can be analyzed. Several end points can be studied, such as histological changes in the mucociliary epithelium, changes in mucus secretion, tumor induction and changes in the in vitro behavior of the epithelial cells after in vivo exposure. In addition, by de-epithelializing the tracheal transplants and reseeding them with another cell population, e.g., from previously treated cell cultures or from human specimens and transplanting them subcutaneously in nude mice, completely new vistas on the effect of chemicals can be opened.

The effects of formaldehyde gas in a flow-through rat tracheal implant system.

A flow-through rat tracheal implant system is described which permits the quantitative and repetitive exposure of respiratory epithelia to test gaseous substances. To investigate the acute toxicity of formaldehyde gas with this new system, open-ended rat tracheal implants were exposed to the gas for 1 h twice a week for up to 8 weeks. The formaldehyde gas was gene-rated from paraformaldehyde, mixed with humidified air and introduced into the lumens of the tracheal implants through microbore tubing. Concentrations of 4.1 +/- 0.9, 8.1 +/- 1.2 or 12.7 +/- 1.5 ppm, were introduced into the trachea by passing an airstream moving at 50, 225 or 475 ml/min, respectively, over the paraformaldehyde heated to 42 degrees C. These formaldehyde concentrations induced mucociliary hypertrophy and increasing amounts of atrophic, transitional and metaplastic epithelia in the tracheal mucosa.

Effects of reduced calcium on proliferation and cell viability in tumorigenic and nontumorigenic rat tracheal epithelial cell lines.

We have compared the in vitro growth and viability of tumorigenic and nontumorigenic rat tracheal epithelial cell lines over a range of calcium concentrations from 0.003 to 0.85 mM. A greater dependence on calcium for proliferation was seen in the nontumorigenic line as compared to the tumorigenic line at both the colony formation level and in mass cultures. In the latter culture condition, a marked differential effect on cell survival was also demonstrated. These differences in calcium dependence were seen in media containing fetal bovine serum or low concentrations of newborn calf serum and in a serum-free medium developed for these cells. The effect was also independent of the method used for calcium removal i.e., either by chelex treatment or the inclusion of EGTA. Therefore, loss of calcium dependence may be associated with tumorigenicity in rat tracheal epithelial cells offering a selectable marker for neoplastic cells in carcinogen-exposed preneoplastic cell populations.