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Effect of Carcinogen Dose on the Dynamics of Neoplastic Development in Rat Tracheal Epithelium¹

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 [M. T., L. R.], and National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [P. N.]

The aim of our studies was to analyze carcinogen dose effects on the development of "carcinogen-altered" preneoplastic epithelial cell populations in rat tracheas following exposure to 7,12-dimethylbenz(a)anthracene. As described previously (Cancer Res., 39: 4003–4010, 1979), cells appear in the tracheal epithelium upon exposure to carcinogen which are distinguishable from the majority of the epithelial cells by a markedly enhanced in vitro growth capacity. In normal tracheal epithelium, most of the clonogenic cells have only a limited replicative capacity. After a limited number of population doublings, tracheal epithelial cell cultures cease to proliferate and senesce. In contrast, cultures established from carcinogenexposed epithelium contain clonogenic cells which proliferate extensively, forming expanding epithelial foci (EF) in late primary cultures. These altered clonogenic units are designated EF-forming units (EFFU). They often escape senescence permanently, thus giving rise to epithelial cell lines. Some of these become neoplastic after repeated subculturing. There are clonogenic units giving rise to subculturable progeny (EFFU_s) and those giving rise to progeny which became anchorage independent (EFFU_s,ag+). In the tracheal cell culture system, anchorage-independent growth is highly correlated with oncogenicity in vivo.

In the present study, tracheas were exposed in vivo for 4 weeks to carcinogen doses ranging from 5 to 335 µg of 7,12'-dimethylbenz(a)anthracene. Tracheal cells were harvested by enzymatic procedures at 0, 4, 16, and 52 weeks after the end of exposure and were assayed in vitro in the epithelial focus assay (EF assay) for the frequency of different types of EFFU. Control tracheas contained less than one EFFU per 10⁵ cells. Exposure to 7,12-dimethylbenz(a)anthracene resulted in a 100- to 500-fold increase. We were unable to detect any definitive carcinogen dose-response effect on the number of clonogenic cells forming EF or subculturable EF (EFFU or EFFU_s), although there was some indication that the latter might be increasing with increasing dose. In contrast, there was a marked carcinogen dose effect on the frequency and rate of appearance of EFFU_s,ag+. For example, at 16 weeks after exposure, the frequencies of EFFU_s,ag+ (as the percentage of all EFFU_s) were 4, 24, and 70% in the tracheas treated with 50, 162, and 335 µg of 7,12-dimethylbenz(a)anthracene, respectively. The data suggest that the carcinogen dose affects either the rate of growth of EFFU_s,ag+, the cell compartment with neoplastic potential, or the conversion rate of EFFU_s to EFFU_s,ag+.

¹ Research sponsored by USPHS Grant Y01-CP-90211 from the Division of Cancer Cause and Prevention, National Cancer Institute, and by the Office of Health and Environmental Research, United States Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.

Received 2/24/82. Accepted 8/ 6/82.