ABSTRACT
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.