Characterization of the purine ring-opened 7-methylguanine and its presistence 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.

8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity.

“…Studies reported here and in the literature point to the identity of 8-hydroxyguanine endonuclease (enzyme A) (3) and FPG protein (6 (33,34) that approximates the planar 8-oxodG lesion.…”

Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA

“…Fapy-7-MeGua has been described in vivo, in the liver of rats treated with N,N-dimethylnitrosamine, or 1,2-dimethylhydrazine, and in rat bladder epithelial DNA after treatment with Nmethylnitrosourea (4,5). Furthermore opening of the imidazole ring of guanine yielding a non-methylated Fapy-Gua has been observed in animal (6) and in human tumor tissues (7).…”

Development and Application of a Sensitive and Rapid Immunoassay for the Quantitation of N7-Methyldeoxyguanosine in DNA Samples

“…Although N7-alkylguanines have been shown to be nonmutagenic per se (15), the N7 substitution on guanine destabilizes the glycosyl bond, resulting in hydrolytic depurination and the generation of apurinic sites or the formation of imidazole ringopened, 5-formamide-4,6-pyrimididine derivatives (16,17). Apurinic sites are promutagenic, promoting the misincorporation of adenine during DNA synthesis if they remain unrepaired (18).…”