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Groups of hairless mice were treated with 4 skin applications of 470 nmol 3-methylcholanthrene (MCA) in benzene and 4 of 20 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) in various sequences, twice a week, together and separately. Three days after the last application, cell kinetic investigations were made comprising the counting of basal and suprabasal cells, the assessment of hyperplasia, the mitotic rate by the stathmokinetic method, the labelling index and the specific activity of DNA after injection of a dose of [3H]dT, and the determination of percentage of cells in each cell-cycle phase by flow cytometry. These studies showed that various treatment schedules with 4 applications stimulated proliferation and caused epidermal hyperplasia, but there was no significant difference between the groups in degree of growth stimulation. There was a significantly higher tumour production by all the combinations than by MCA alone. It was of no significant importance for the tumour production whether the 4 applications of MCA came before or after the 4 of TPA. Alternating treatment (MCA-TPA, etc.) seemed to give a higher tumour risk than the other treatment sequences. The consequences of these results for the two-stage theory of carcinogenesis (stating that initiation must come first) are discussed, and it is concluded that (at least under the experimental conditions used here) initiation does not need to come first for a good tumour yield.

Must initiators come first? Tumorigenic and carcinogenic effects on skin of 3-methylcholanthrene and TPA in various sequences