The time needed for normalization of hairless mouse epidermis after treatment with twice weekly topical skin applications of 10 nmol 12-O-tetradecanoylphorbol-13-acetate in acetone, or acetone alone, for 18 weeks. A morphologic and cell kinetic study

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Abstract
In order to find the time needed for normalization of the mouse epidermis after an 18 wk-course of treatment according to the 2-stage carcinogenesis protocol, hairless mice were treated with 10 nmol 12-O-tetradecanoylphorbol-13-acetate in 100 .mu.l reagent grade acetone twice weekly for 18 wk. A control group was treated similarly with 100 .mu.l reagent grade acetone alone. Groups of 4 animals were killed each week for 10 wk thereafter, and morphologic and cell kinetic studies performed. To assess hyperplasia, the number of basal and subrabasal cells per unit length of epidermis was counted. The mitotic rate was determined by a stathmokinetic method using colcemid for arrest of mitoses. DNA synthesis was measured by determining the labeling index and the DNA-specific activity after injection of [3H]thymidine. Flow cytometric measurements were performed to find the fraction of cells in the various cell cycle phases at each observation point. Treatment with acetone alone led to a moderate hyperplasia and a slight increase in the mitotic rate, which persisted during the 10 wk of subsequent observation. Treatment with TPA led to a considerable hyperplasia which first increased for 3 wk and then decreased, and was still significant 10 wk after termination of treatment. The line of best fit through the values indicated that a time of .apprx. 20 wk may be needed before normal epidermal thickness is reestablished. TPA treatment also led to a pronounced increase in the labeling index and a high mitotic rate. These variables became almost normal after .apprx. 8-10 wk, but one cannot know whether one or more new waves of proliferation take place > 10 wk after termination of TPA treatment. If a proper inverse experiment according to the 2-stage theory of carcinogenesis should be performed, one had to wait at least 20 wk after termination of promotion before the initiating dose of carcinogen should be applied. The possibility of increased acquired resistance to carcinogens during promotion is also discussed.