In vivo formation of oxidized DNA bases in tumor promoter-treated mouse skin.

There has been a paucity of evidence showing that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter, causes DNA damage in vivo. We show that oxidized DNA bases are formed in the epidermis of TPA-treated SENCAR mice in a dose- and time-dependent manner. As measured by high-performance liquid chromatography and acetylation of nucleosides with [3H]acetic anhydride, these oxidized DNA derivatives include cis-thymidine glycol, 5-hydroxymethyl-2'-deoxyuridine, and 8-hydroxyl-2'-deoxyguanosine. Their maximal formation induced by a single TPA dose occurred within 6-8 h (a 2-5-fold increase). The level of 8-hydroxyl-2'-deoxyguanosine was the lowest (3.2/10(5) bases) and remained almost unchanged for 18 h; thymidine glycol (29.1/10(4) bases) and 5-hydroxymethyl-2'-deoxyuridine (17.3/10(4) bases) declined gradually but were still above controls at 24 h. Reapplication of TPA 20 h after the first dose (time of the maximal polymorphonuclear leukocyte infiltration) enhanced the net formation of 8-hydroxyl-2'-deoxyguanosine by 3.8-fold (P less than 0.05), of cis-thymidine glycol by 1.9-fold (P less than 0.001), and of 5-hydroxymethyl-2'-deoxyuridine by 2.0-fold (P less than 0.01), as compared to those maximally produced by a single TPA dose. Thus, the infiltration of polymorphonuclear leukocytes into TPA-treated mouse skin, which was corroborated by histological examination and the presence of polymorphonuclear leukocyte-specific myeloperoxidase, might play an important role in TPA-induced DNA oxidation in vivo. Our findings provide proof that tumor promoters can induce genetic modification in vivo that is oxidative in nature. Hence, formation of oxidized DNA bases may be responsible for the genetic effects of tumor promoters in carcinogenesis.