Estrous Cycle Modification of Rat Mammary Gland DNA A1kylation by TV-Methyk/V-nitrosourea1

Virgin Sprague-Dawley rats exhibiting regular estrous cycles were used as a model system to determine whether the level of circulating estrogen modifies the alkylation pattern of mammary gland DNA by a direct-acting carcinogen, /V-methyl-iV-nitrosourea (NMU). The concen tration of 7-methylguanine and O'-methylguanine were similar in mam mary epithelial DNA 0.2S, 0.50, and 1.0 h after i.v. injection of 50 mg/ kg body weight NMU on different days of the rat estrous cycle. However, 0Â°-methylguanine was significantly higher in mammary gland DNA 8 and 24 h after a single i.v. dose of carcinogen on proestrus or estrus, compared to rats receiving carcinogen on diestrus. There was no differ ence in the 7-methylguanine levels at 8 h in any group, but this adduct was higher in estrous-lreated rats at 24 h. The ratio of O6-methylguanine to 7-melhylguanine was significantly lower at 8 h in mammary gland DNA from diestrous-injected rats, and this difference reflected the lower level of O'-methylguanine adducts in this group. In contrast, 0*-methylguanine concentrations in DNA extracted from the liver of the same animals were virtually identical at all time periods examined. 7-Methylguanine levels were higher in the liver at 0.5, 1, 8, and 24 h post-NMU in proestrus as compared with diestrous-injected rats. The observed adduct clearance suggests that rat mammary epithelium may contain repair systems capable of removing O'-methylguanine. These results also suggest that the initial removal of the O6-methylguanine lesions in mammary epithelial DNA (rather than the initial rate of alkylation) is affected by the hormonal environment during carcinogen exposure. This effect may be tissue specific since removal of 06-methylguanine from liver DNA is apparently not altered by the stage of the estrous cycle at which NMU is administered. scriptum begins within 2 h and reaches a peak within 24 h following a rise in circulating estrogen (6). The induction of tumors by alkylating agents such as DMBA or NMU is asso ciated with the chemical modification of cellular DNA (7). Unlike DMBA (7), DNA adduct formation following NMU administration occurs rapidly (8). The short half-life (<10 min) of NMU (9) and rapid rate of adduct formation suggested that we could investigate whether the physiological amount of estrogen(s) present at the time of carcinogen exposure influenced mammary tumorigenesis by quantitatively or qualitatively al tering the initial rate of alkylation or removal of the resulting lesions in mammary gland DNA. Although NMU is capable of producing a large number of lesions in DNA by reacting with all nitrogens and oxygens in both pyrimidines and purines, a primary lesion implicated in alkylation carcinogenesis is O6-meGua (10-17) rather than 7 meGua, the predominant adduct (18). For example, induction of thymic lymphomas in CS7BL mice by a variety of alkylating agents correlated well with alkylation at the Opposition of guanine (19). Accumulation and persistence of O6-meGua has also been implicated in the neurooncogenicity of NMU (8). The present communication describes the results of studies in which the formation and persistence of O6-meGua and 7-meGua in mammary gland and liver DNA were quantitated following exposure of cycling female rats to a single i.v. 50-mg/kg body