David G. Thomassen

QUANTITATION OF CELL PROLIFERATION, COLONY FORMATION, AND CARCINOGEN INDUCED CYTOTOXICITY OF RAT TRACHEAL EPITHELIAL CELLS GROWN IN CULTURE ON 3T3 FEEDER LAYERS

SummaryA cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.

Hapten carrier conjugates associated with halothane hepatitis.

The elucidation of the mechanism of hepatitis caused by the inhalation anesthetic agent halothane (CF3CHClBr) (Satoh et al., 1987; Neuberger and Kenna, 1987; Weis and Engelhardt, 1989) is important since halothane is still widely used in adults in the United Kingdom and Europe (Neuberger and Kenna, 1987; Weis and Engelhardt, 1989) and is often an agent of choice for children (Hals et al., 1986; Whitburn and Sumner, 1986; Kenna et al., 1987a). Also, recent reports suggest that the structurally related inhalation anesthetic enflurane (CHF2OCF2CHFCl), which is widely used in adults in the United States, may cause liver damage by a similar process (Christ et al., 1988a; Christ et al., 1988b).

ROLE OF GENE AND CHROMOSOMAL MUTATIONS IN CELL TRANSFORMATION

To understand the role of mutagenesis in carcinogenesis fully, we must consider all types of mutations including gene, chromosomal, and gene-number mutations and all changes involved in the progressive development of neoplastic cells. We have found that certain known human carcinogens (i.e., DES and asbestos), which were classified as epigenetic carcinogens based on gene-mutation assays, have mutational activity at the chromosomal level that correlates with their ability to induce cell transformation. This should caution against classification of chemicals as genotoxic or epigenetic without a complete understanding of their mechanism of action. Furthermore, our studies indicate that more than gene-mutation assays is needed for carcinogen testing. In particular, chromosomal changes induced by chemicals, both aberrations and aneuploidy, need to be carefully assessed. In addition, the role of all types of mutation in the overall process of neoplastic transformation needs to be determined. This can only be examined by studying each individual change involved in neoplastic progression. Thus, any attempt to assess a chemicals carcinogenic potential should consider not only all types of mutational changes but both early and late changes involved in neoplastic transformation.

Variable responsiveness of rat tracheal epithelial cells to bovine serum albumin in serum-free culture

SummaryThe colony-forming efficiency of rat tracheal epithelial (RTE) cells was determined in serum-free media containing different types of commercially available bovine serum albumin (BSA): crude fraction V, essentially globulin-free, essentially fatty-acid-free, and essentially globulin- and fatty-acid-free BSA. RTE cells exhibited a concentration-dependent increase in colony-forming efficiency in response to crude fraction V BSA. Similar results were obtained using essentially globulin-free BSA. However, deletion of cholera toxin from the medium resulted in a decrease in the colony-forming efficiency for cells plated in high concentrations (>2 mg/ml) of globulin-free, but not one type of fraction V, BSA. Essentially fatty-acid-free or essentially fatty-acid- and globulin-free BSA stimulated RTE cell colony formation at low concentrations (less than 2.5 to 5 mg BSA/ml) but resulted in concentration-dependent decreases in colony-forming efficiency at higher concentrations. The response of cells to these BSAs was not dependent on cholera toxin. Finally, commerically available fraction V BSA prepared by heat shock, dialysis, charcoal treatment, and deionization was stimulatory at low concentrations but inhibitory at high concentrations. These data suggest that impure preparations of BSA can, under different conditions, stimulate or inhibit cell proliferation and that the expression, of these activities is affected by the method of BSA preparation, the concentration of BSA used, and, in some cases, by the presence or absence of cholera toxin.

Effect of Stress on Drug Hypersensitivity

The phenomenon of drug allergies has long puzzled medical practitioners and scientists alike. As a highly individual response it represents an unpredictable factor in drug development, which may not be noticed until several years after a new drug has been released for use in the general population. To date, it is not known what predisposes an individual to develop hypersensitivity to a certain drug. The focus of this article is to examine the role of stress as a possible contributing factor in drug hypersensitivity

A Quantitative, Clonal Assay for Carcinogen-Induced Alterations of Respiratory Epithelial Cells in Culture

The identification of environmental carcinogens and the assessment of the potential risks of these substances to humans require experimental systems to measure quantitatively the activity of carcinogens and promoters. Cell culture systems are potentially very useful experimental models for such studies. Short-term, inexpensive cell culture assays for carcinogens are available. These assays have a high predictive ability for the detection of known carcinogens with few false positive results (Barrett et al., 1980). The end point measured, preneoplastic or neoplastic transformation of the test cells, is relevant to the carcinogenic process and is not predicated on a theoretical correlation. These systems can also be used for mechanistic studies on the cellular and molecular basis of neoplastic development. The results with cells and tissues of different species (including human) can be compared and contrasted under similar experimental conditions.

QUANTITATIVE, CLONAL STUDIES OF NEOPLASTIC PROGRESSION OF RAT TRACHEAL EPITHELIAL CELLS

We have developed a system for quantitating early carcinogen-induced changes of rat tracheal epithelial (RTE) cells and their subsequent progression to neoplasia.’” Methods are described for growing normal RTE cells in culture and for selecting and quantitating the appearance of enhanced growth (EG) variants. Normal RTE cells were isolated by pronase treatment of Fischer 344 rat tracheas and grown on lethally irradiated 3T3 cells in Ham’s F12 medium with 5% fetal bovine serum, insulin (1 Clg/ml), and hydrocortisone (0.1 fig/ml). Colony-forming efficiencies were 5-10% for at least 5 passages (>20 population doublings). N-Methyl-N’-nitro-N-nitrosoguanidine (MNNG) treatment resulted in a dose-dependent reduction in colony-forming efficiency. One to seven days after MNNG treatment, EG variants were selected by removing feeder cells and refeeding cultures with complete medium or by replating RTE cells onto dishes without feeders. After four weeks, large colonies of small hyperchromatic cells with increased nuclear-cytoplasmic ratios were observed. Treatment of RTE cells with MNNG also resulted in a dose-dependent increase in the frequency of EG variants. The maximum transformation frequency in the treated cultures (3.7% per colony-forming cell) was 100-fold greater than in control cultures when selection for EG variants was applied seven days after treatment. The dose response of MNNG-induced transformation was linear with a slope of 1.09 when plotted as the logarithm of cell-transformation frequency versus the logarithm of dose, which is consistent with a one-hit phenomenon. Similar results were obtained with a wide variety of carcinogens including y radiation, benzo[a]pyrene, benzo[a] pyrene diol epoxide, and nickel salts. The frequency of carcinogen-induced preneoplastic RTE cells was calculated based on the Poisson distribution (Po) method from the fraction of dishes without transformed colonies. The distribution of EG variants statistically followed a Poisson distribution and allowed the use of this method for calculation of the transformation frequency. In addition, comparable results were obtained when the transformation frequencies were calculated based on the actual number of variants obtained. EG variants could be subcultured, and about 50% formed permanent cell lines. Continued propagation of cultures of EG variants resulted in the accumulation of cells capable of growing in semisolid agarose medium. These colonies when isolafed had high colony-forming efficiencies in agarose medium (540%) and were nontumorigenic in nude mice. After extensive propagation in vitro, EG variants developed neoplastic potential and formed squamous cell carcinomas when injected into nude mice. This work provides a system for characterizing and quantitating the frequency of early carcinogen-induced changes and the development of neoplasia in respiratory epithelial cells.