No abstract

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.

Hepatic N-oxidation, followed by N-glucuronidation, has been proposed as a route of metabolic activation for arylamine bladder carcinogens. It is postulated that the N-glucuronides are transported to the bladder lumen where they are hydrolyzed under slightly acidic conditions to release directacting carcinogenic and mutagenic N-hydroxyarylamines. In this study, 4-aminobiphenyl (ABP), 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), 2-acetylaminofluorene (AAF), 4-nitrobiphenyl (NBP), benzidine (BZ), and N-acetylbenzidine (ABZ) were administered to male beagle dogs (60 mmole/kg), and the bladder epithelium DNA adducts were quantified at various times after treatment. At 24-48 hr after administration, the order of binding to bladder epithelium DNA was: ABP >> AAF > NBP 2-NA-BZ ABZ>> 1-NA. The level of DNA modification by ABP remained constant for 7 days, whereas 2-NA and AAF residues decreased by 35% and 80%, respectively. The extent and relative persistence of total DNA binding correlated with the compounds' ability to induce bladder tumors in dogs. ABP, AAF, NBP, 2-NA and ABZ administration resulted in DNA binding sufficient for adduct analysis. Enzymatic hydrolysis of the DNA and examination of the adducts by high pressure liquid chromatography indicated that arylamine substitution at C8 of deoxyguanosine was the dominant product. Additional adducts were detected in animals treated with ABP, NBP, and 2-NA. Furthermore, the profiles of adducts obtained in vivo were remarkably similar to the profiles obtained when the N-hydroxy arylamine metabolites of these carcinogens were reacted with DNA in vitro at pH 5.0. To evaluate the mutagenic potential of these arylamine-DNA adducts, Salmonella typhimurium strains TA 1535 and TA 1538 were incubated with N-hydroxy-2-NA, N-hydroxy-2-aminofluorene (AF), N-hydroxy-ABP, and N-hydroxy-ABZ and the resulting DNA adducts and reversions were quantified. Arylamine-C8-deoxyguanosine substitution was correlated with frameshift reversions induced by these agents, with the lesions showing a relative order of mutagenic efficiency of ABZ>AF-2-NA>ABP. These data suggest that mutagenic N-hydroxyarylamines may be ultimate carcinogens for the bladder epithelium. Furthermore, if one assumes that a mutagenic lesion is important for tumor initiation, then C8-deoxyguanosine substitution by these compounds may be significant for urinary bladder carcinogenesis.

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.

A major and previously undetected carcinogen-DNA adduct was found in the livers of rats given N,N-dimethylnitrosamine or 1,2-dimethylhydrazine. This adduct, which accounted for 55% of the total methyl residues in DNA at 72 hours after carcinogen treatment, was chromatographically identical to a synthetic purine ring-opened derivative of 7-methylguanine and could be released from the isolated hepatic DNA by a specific E. coli glycosylase. The synthetic ring-opened adduct was characterized by mass and NMR spectroscopy as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine and appears to exist in two rotameric forms.