Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O2-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O2-alkylcytosines, 3-, O6-, and 7- alkylguanines and O2-, 3-, and O4-alkylthymines. N6-Alkyladenines, 1-alkylguanines and N2-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [14C]methyl methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.

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Purine ring-opened 7-methylguanine, prepared in vitro by alkaline treatment of 7-methylguanosine or of methylated calf thymus DNA, was extensively characterized by chromatographic and spectral techniques as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine. This modified base chromatographed as an early-eluting peak on an ion-exchange column but separated into two interconvertible components after reversed-phase or porous-resin h.p.l.c. The two components were analyzed by thermal desorption mass spectrometry and 500 MHz 1H-n.m.r. spectroscopy. Their mass spectra were identical (M+ at m/z 183) and their n.m.r. spectra each exhibited the same two sets of resonances whose relative intensities were solvent-dependent. Analysis by h.p.l.c. showed interconversion of the two components and kinetic studies demonstrated that this reaction was a reversible first-order process. At equilibrium, k1 = k2 = 0.334 h-1 and delta G = 22.9 kcal/mol. These data indicated that the ring-opened 7-methylguanine exists as cis/trans isomers with restricted rotation about the amide bond. Treatment of rats with an intraurethral initiating dose of the carcinogen N-methylnitrosourea resulted in a high level of bladder epithelial DNA modification with 7-methylguanine, O6-methylguanine, and methyl phosphotriesters as major adducts at 2 h after instillation. Purine ring-opened 7-methylguanine, chromatographically identical to the in vitro products, was initially a minor adduct. However, it was the only persistent modification in the bladder epithelial DNA and eventually accounted for 72% of the total carcinogen binding after 21 days. A tumor-promoting regimen, involving dietary sodium saccharin, did not alter the repair or persistence of any of the methylated adducts. These data demonstrate that purine ring-opened 7-methylguanine, previously reported to exist in liver DNA after N,N-dimethylnitrosamine or 1,2-dimethylhydrazine treatment, is present in a carcinogen-target tissue and is considerably more persistent than O6-methylguanine or other DNA methylation products. The possible role of this adduct as a promutagenic lesion initiating urinary bladder carcinogenesis is discussed.

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Genistein and ethinyl estradiol (EE 2 ) were examined in multigenerational reproductive and chronic toxicity studies that had different treatment intervals among generations. Sprague-Dawley rats received genistein (0, 5, 100, or 500 ppm) or EE 2 (0, 2, 10, or 50 ppb) in a low phytoestrogen diet. Nonneoplastic effects in females are summarized here. Genistein at 500 ppm and EE 2 at 50 ppb produced similar effects in continuously exposed rats, including decreased body weights, accelerated vaginal opening, and altered estrous cycles in young animals. At the high dose, anogenital distance was subtly affected by both compounds, and a reduction in litter size was evident in genistein-treated animals. Genistein at 500 ppm induced an early onset of aberrant cycles relative to controls in the chronic studies. EE 2 significantly increased the incidence of uterine lesions (atypical focal hyperplasia and squamous metaplasia). These compound-specific effects appeared to be enhanced in the offspring of prior exposed generations.