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Increased in Vitro Growth Capacity of Tracheal Epithelium Exposed in Vivo to 7,12-Dimethylbenz(a)anthracene¹

Cancer and Toxicology Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Heterotopic tracheal transplants of rats were exposed in vivo to 150 or 640 µg of 7,12-dimethylbenz(a)anthracene (DMBA) delivered over 2 weeks, and the epithelium was studied in vitro in an attempt to identify changes in growth behavior during early phases of neoplastic development. Explants were made from the exposed tracheas, as well as from a series of controls, and the rate of epithelial outgrowth from the explants was measured. Secondly, the capacity of the outgrowths to survive as primary cultures and their ability to survive multiple in vitro passages were studied.

During the initial planting, the rate of outgrowth was by far the greatest from the explants preexposed to 150 µg DMBA. Outgrowth from explants preexposed to 640 µg DMBA was sparse during the first planting but increased markedly during repeated planting when insulin- and hydrocortisone-supplemented medium was used. Establishment of outgrowth during repeated planting of carcinogen-exposed tracheal pieces was clearly hormone dependent. Control explants did not exhibit this hormone dependency.

Primary cultures could be established from only three of six explants preexposed to 150 µg DMBA. These explants had been initiated in insulin- and hydrocortisone-supplemented medium. The primary cultures were successfully subcultured. Primary cultures were also established from five of five explants preexposed to 640 µg DMBA and cultured in hormone-supplemented medium. At least one cell line was obtained from each of the explants. To establish and maintain primary cultures from control tracheas required an enriched medium containing amino acid supplements, sodium pyruvate, and putrescine, as well as the hormone supplements. However, such cultures could not be subcultured. The primary cultures from the carcinogen-exposed explants and the subsequently developed cell lines all exhibited morphological characteristics of keratinizing squamous epithelium. These characteristics include: epithelioid cell morphology, multilayering and sloughing of orangeophilic squamous cells, and the presence of keratohyalin granules.

These experiments demonstrate a markedly increased in vitro growth capacity of tracheal epithelium after a short in vivo exposure to carcinogen.

¹ Research jointly sponsored by the Carcinogenesis Program of the National Cancer Institute, the Environmental Protection Agency, and the Energy Research and Development Administration under contract with the Union Carbide Corporation. By acceptance of this article, the publisher or recipient acknowledges the right of the U. S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

² To whom requests for reprints should be addressed.

Received 12/21/76. Accepted 3/11/77.