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Inhibition by 2,6-Dithiopurine and Thiopurinol of Binding of a Benzo(a)pyrene Diol Epoxide to DNA in Mouse Epidermis and of the Initiation Phase of Two-Stage Tumorigenesis¹

Department of Carcinogenesis, Science Park-Research Division, University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957

The chemotherapeutic agent 6-mercaptopurine was previously shown to inhibit the binding of 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydro-benzo(a)pyrene (BPDE-I) to DNA in Chinese hamster ovary cells. Two compounds related to 6-mercaptopurine, 2,6-dithiopurine (DTP) and thiopurinol (TP), have been tested for inhibition of the binding of BPDE-I to epidermal DNA in mouse skin. Doses of test compound (0.2–20 µmol) or solvent control were applied to the shaved backs of female SENCAR mice. Fifteen min later, 200 nmol [³H]BPDE-I were applied to the same area and 3 h later the mice were sacrificed and epidermal DNA was purified and adduct formation was quantitated radiometrically. At the highest doses studied, DTP and TP inhibited DNA binding by 90 and >80%, respectively. The dose necessary to inhibit DNA binding by 50% was about 0.8 µmol for DTP and about 2 µmol for TP. To test whether this protective effect was long-lasting, the time between application of purinethiol and [³H]BPDE-I was systematically increased. Although the level of protection was decreased by increasing the time between applications, both compounds inhibited binding 50–60% even after 24–48 h. A radioactive compound tentatively identified as a TP-BPDE-I adduct could be recovered from epidermal homogenates following topical application of TP and BPDE-I. We used a standard two-stage initiation-promotion protocol to test the effects of these compounds on mouse skin carcinogenesis. Mice were treated with 0, 1, or 10 µmol of either TP or DTP, and 15 min later were treated with an initiating dose of BPDE-I (200 nmol). Twice weekly promotion with 12-O-tetradecanoylphorbol-13-acetate was begun 2 weeks later and continued for 23 weeks. A dose-dependent inhibition of tumor incidence and multiplicity was noted with both compounds. Treatment of skin with 10 µmol of DTP prior to initiation lowered the number of papillomas per mouse by >90% compared to solvent controls; a 10-fold lower dose resulted in about 50% inhibition. The 10-µmol dose of TP resulted in about 50% inhibition. Mice were examined for 50 weeks for the presence of squamous cell carcinomas. Compared to the positive control group, 10 µmol DTP inhibited carcinoma incidence and lowered the total number of carcinomas by 90–95%. Treatment with 10 µmol TP had no significant effect on carcinoma incidence, and only slightly lowered the total number of carcinomas.

¹ This research was supported by Grant RD-299 from the American Cancer Society and Grant CA 40823 from the NIH.

² To whom requests for reprints should be addressed.

Received 4/26/91. Accepted 7/ 8/91.