title: Characterization of the purine ring-opened 7-methylguanine and its persistence in rat bladder epithelial DNA after treatment with the carcinogen N-methylnitrosourea.
abstract: 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.
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification*; D004848:Epithelium / Q000187:drug effects; D005260:Female; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008770:Methylnitrosourea / Q000378:metabolism / Q000633:toxicity*; D009607:Nitrosourea Compounds / Q000633:toxicity*; D011743:Pyrimidines / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium; D001743:Urinary Bladder / Q000187:drug effects*
chemical_list: D009607:Nitrosourea Compounds; D011743:Pyrimidines; D014316:Tritium; D008770:Methylnitrosourea; C037301:N(5)-methyl-N(5)-formyl-2,5,6-triamino-4-hydroxypyrimidine; D004247:DNA