There is increasing evidence that tumor promoters may act, at least in part, by induction of active oxygen species' which are capable of oxidizing DNA bases. We found that there is a correlation between the in vivo first-stage tumor promoting activity, production of H2O2 by promoter-stimulated human polymorphonuclear leukocytes (PMNs) and formation of oxidized bases in DNA exposed to them. Iron ions present in a complex with EDTA or with transferrin were needed for the formation of 5-hydroxymethyl-2'-deoxyuridine (HMdU) and thymidine glycol (dTG). Autologous plasma substantially potentiated DNA damage. 12-0-tetradecanoyl-phorbol-13-acetate (TPA) also caused formation of HMdU in the DNA of HeLa cells, the amount of which was increased in the presence of PMNs. One of the main objectives of this proposal is to determine whether there is also a correlation between the in vivo first-stage promoting potency and formation of HMdU, dTG and 8-hydroxy-2'-deoxyguanosine (80HdG) in the DNA of HeLa cells, as well as in Bloom's syndrome and normal human fibroblasts. The in vivo formation of oxidized bases will be tested by the application of TPA, mezerein (Mez) and 12-0-retinoyl-phorbol-13-acetate (RPA) to mouse skin (SENCAR and C57BL/6). DNA will be isolated from the epidermal cells, digested to 2'-deoxyribonucleosides, and analyzed by HPLC for HMdU, dTG and 80HdG. The second objective is to determine whether the promotional effects of complete carcinogens that require oxidative metabolism also involve formation of active oxygen species and oxidized bases. We have found that incubation of rat liver microsomes with benzo(a)pyrene (B(a)P) causes a dose-dependent production of H2O2 and formation of HMdU and dTG in co-incubated DNA that is inhibited by catalase. The non-carcinogenic PAHs pyrene and anthracene were inactive. To achieve this objective, the effects of carcinogens (B(a)P, 7,12-dimethyl-benz(a)anthracene and 5- methylchrysene) and co-carcinogens (benzo(e)pyrene and 6- methylchrysene) on formation of H2O2 and of oxidized bases in DNA will be tested in various systems. These include incubations with induced and uninduced mouse liver microsomes (same mouse strains as above), with HeLa cells with and without microsomes, and treatment of mouse skin with carcinogenic PAHs. Formation and persistance of oxidized bases in DNA of PAH-treated mouse skin will be compared to those resulting from treatment with tumor promoters.
Showing the most recent 10 out of 35 publications
