Two-stage carcinogenesis with rat embryo cells in tissue culture.

Transformation of rat embryo fibroblasts in vitro has been investigated using initiation with either benzo(a)pyrene (BaP), 7,12-dimethylbena(a)anthracent (DMBA) or benzo(e)pyrene (BeP) and promotion with either phorbol ester (TPA) or croton oil (Cr.Oil). The criteria used to assess in vitro transformation were (a) the efficiency of cloning in liquid medium, (b) abnormal cellular morphology and (c) the development of malignant tumours following s.c. inoculation of newborn rats. The results show that the cloning efficiency, which remained low in the control cells, was increased to a variable extent in the treated groups. Transformation occurred in all groups, but occurred earliest in cells that were initiated and promoted. Initiation with DMBA or BaP and promotion with TPA or Cr.Oil led to the earliest acquisition of malignancy. Correlations were found between the transformation of cells in vitro and the acquisition of malignant potential, and between the carcinogenic action of the compounds in vitro and their action in vivo, but cloning efficiency was not a reliable indicator of in vitro transformation or of malignancy. In most cases in vitro transformation appeared to precede the acquisition of malignancy, but in two cases it occurred later. The studies also show that BeP, which is a tumour initiator in vivo, also acts in this way in vitro. The conclusion drawn from a discussion of these results and of two-stage carcinogenesis in vivo is that two-stage carcinogenesis can be reproduced in tissue culture; this model may be useful in studies of those mechanisms of chemical carcinogenesis that involve the processes of initiation and promotion

Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene.

Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene