In order to complete our studies of factors influencing the initiation of hepatocarcinogenesis, the survival of damaged hepatocytes must be enumerated. The yield of hepatocytic neoplasms is the product of the frequency of induced events per survivor and the surviving fraction. Cell cycle-dependent variation in survival must be factored into the results of the previous studies in which yields of neoplasms were enumerated. Several methods will be applied to assess cell survival after carcinogen treatment including in situ staining with trypan blue dye and colonization of fat pads by isolated hepatocytes. With an estimate of cytotoxicity the frequencies with which hepatocarcinogens initiate carcinogenesis can be determined. With an improved measure of initiation frequency we can quantify the effects of different carcinogens at equitoxic doses. We have found that three carcinogens that produce quite different spectra of lesions in DNA display differences in their abilities to induce islands of cellular alteration and neoplasms. At doses of the carcinogens producing nearly equal numbers of islands, the methylating agent, DMN-Ac, produced multiple neoplasms in every liver, the arylalkylating agent, BPDE, produced a few neoplasms with moderate incidence, and the physical carcinogen, gamma radiation, produced no tumors. We will establish for each of the three carcinogens quantitative dose-response curves for frequencies of carcinogenic initiations and cellular alterations in sensitive and resistant populations. Because of the possibility of variable latencies for neoplasms initiated by the three carcinogens we also will perform time-course studies to determine initiation and alteration frequencies after a single dose given to sensitive populations. Finally we will establish the frequencies and timing of appearance of cell populations which express an altered oncogene product or aneuploidy. Thus we will determine which alterations are seen in livers that have low probability to develop neoplasms and which are seen in livers with high probability. In this way we will begin to define the specific alterations that provoke the neoplastic phenotype in hepatocytes that have been damaged when in sensitive or resistant cell cycle phases.
