methylnitronitrosoguanidine and 7-methylguanine

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. (1)H and (13)C NMR chemical shifts of 5-formyl-2'-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol. Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

Topics: Adenine; Base Pair Mismatch; DNA Damage; DNA Glycosylases; DNA Repair; Escherichia coli; Guanine; Kinetics; Magnetic Resonance Spectroscopy; Methylnitronitrosoguanidine; Molecular Structure; N-Glycosyl Hydrolases; Oligodeoxyribonucleotides; Pentoxyl; Substrate Specificity; Thymine; Uracil

A hybridoma (K1A8) secreting a high affinity antibody to imidazole ring-opened 7-methylguanine (N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine) was obtained from spleen cells of a mouse immunized with a conjugate of keyhole-limpet hemocyanin and imidazole ring-opened 7-methylguanylic acid (iro-7mGMP). The antibody recognizes the iro-7-methylguanine (iro-7mG) determinant in the BSA-iro-7mGMP conjugate, in chemically methylated, denatured DNA, and in the ring-opened 7-methylguanosine, 7-methyldeoxyguanosine and 7mGMP haptens. In the competitive ELISA of DNA-iro-7mG, 50% inhibition (I50) was observed at 4 fmol determinant per well (8 x 10(-11) M) using BSA-iro-7mGMP as the immobilized antigen. The lower limit of 7-methylguanine (7mG) detection in DNA is determined by the binding of unmodified DNA per se to the antibody. The intrinsic reaction of DNA with antibody is low; in the competitive ELISA I50 was obtained with 330 micrograms calf thymus DNA per 50 microliters well, equivalent to 4 nmol iro-7mG per mol nucleotide. The 7mG content of calf thymus DNA is 7 nmol per mol nucleotide (approximately 20 amol per micrograms DNA). The limit of detection of 7mG by competitive ELISA is quoted provisionally as 7 nmol iro-7mG per mol nucleotide, where 30% inhibition of antibody binding is obtained in the presence of 105 micrograms DNA per 50 microliters well. Nuclear DNAs of tissue culture cells treated with 0, 0.01 and 0.1 mM N-methyl-N'-nitro-N-nitrosoguanidine contained 0.18, 31 and 320 mumol, respectively, of 7-methylguanine adducts per mol of nucleotides. This report indicates that the K1A8 antibody will serve to quantify DNA alkylation in human populations exposed to low levels of methylating carcinogens.

Topics: Alkylating Agents; Alkylation; Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Cells, Cultured; DNA; Enzyme-Linked Immunosorbent Assay; Epitopes; Guanine; Haptens; Hybridomas; Lymphocytes; Methylnitronitrosoguanidine; Mice; Pyrimidines; Sulfuric Acid Esters

We have isolated and characterized a mutant of baker's yeast, Saccharomyces cerevisiae, carrying the new mutation, ngs1, which is sensitive to the toxic effects of monofunctional alkylating agents, but normal with respect to 254-nm ultraviolet light sensitivity. ngs1 mutants exhibited more or less normal reversion frequencies for his1-7 and ilv1-92 induced by each of these mutagens. The various sensitivities associated with ngs1 cosegregated and have been shown to be the result of a lesion in a single nuclear gene. Extracts of ngs1 and NGS1+ strains contained approximately equal levels of an activity that removes 3-methyladenine (3MA) and 7-methylguanine (7MG) from DNA in vitro. The mutation also depressed sporulation.

Topics: Adenine; Alkylating Agents; DNA Repair; Genes, Fungal; Guanine; Methylnitronitrosoguanidine; Mutation; Saccharomyces cerevisiae; Sulfuric Acid Esters

Topics: Administration, Oral; Animals; Duodenal Neoplasms; Guanine; Jejunal Neoplasms; Methylnitronitrosoguanidine; Rats; Rats, Inbred Strains; Stomach Neoplasms

Salmonella tester strains which are reverted by base-pair substitution mutagens are relatively insensitive to the mutagenic effects of N-methyl-N-nitroso compounds. One reason for this insensitivity is the ability of these strains to withstand low doses of these compounds before they become sensitive to their mutagenic effects. In this report it is shown that mutagenesis induced by treatment of Salmonella typhimurium TA 1535 with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) in buffer is biphasic with a low sensitivity range at low doses where little mutagenesis occurs, followed by a high sensitivity range whose onset begins after an apparent threshold dose has been exceeded. levels of O6-methylguanine (O6-MeG) in the DNA extracted from the bacteria follow a similar dose-response curve suggesting a dependency of mutagenesis on O6-MeG. In contrast, levels of 7-methylguanine (7-MeG) in the DNA increase linearly with dose. O6-MeG was undetectable at the lowest dose of MNNG whereas 7-MeG was readily detectable. Although such resistance to O6-alkylation has been demonstrated in MNNG- pretreated (adapted) E. coli, it has not been reported in unpretreated cells. Then isolated DNA was treated with MNNG a linear dose-response in the generation of O6-MeG was observed. The lack of O6-MeG in DNA isolated from MNNG treated cells after low doses is attributed to a saturable, constitutive repair activity in the bacteria. An attempt to observe the removal of O6-MeG from the bacteria after exposure to a short challenge dose of N-nitroso-N-methylurea (NMU) followed by a subsequent incubation in buffer was unsuccessful, probably because all the repair occurred within the time necessary to treat and lyse the cells.

Topics: Chromatography, High Pressure Liquid; DNA; DNA Repair; Dose-Response Relationship, Drug; Guanine; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutation; Salmonella typhimurium; Time Factors

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been reported to induce BHK-21/Cl 13 cell growth in agar suspension. To determine if MNNG was also mutagenic to BHK cells, an ouabain-resistance mutation assay was established using these cells. In this system MNNG was compared to nitrosocimetidine (NC). MNNG and NC did induce ouabain-resistant mutations in BHK cells. The ability of the test compounds to methylate DNA in BHK cells was also determined, and both MNNG and NC yielded detectable levels of 7-methylguanine in treated cells. MNNG and NC were tested for the ability to transform BHK cells, and did. NC was found to be as effective a mutagen and transforming agent in BHK cells as MNNG when administered at equitoxic concentrations; approx. 4-fold less effective at equimolar concentrations.

Topics: Animals; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Cimetidine; Cricetinae; DNA; Guanidines; Guanine; Kidney; Mesocricetus; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens

Topics: 1,2-Dimethylhydrazine; Adenine; Alkylating Agents; Animals; Carcinogens; Dimethylhydrazines; Dimethylnitrosamine; Guanine; Male; Methyl Methanesulfonate; Methylation; Methylnitronitrosoguanidine; Niacinamide; Rats