methylnitronitrosoguanidine and 6-ethylguanine

The biological effects of DNA adducts depend on their nature, and on their half-lives relative to the rates of DNA replication and transcription. Their half-lives are determined by the rates of spontaneous decay, such as depurination, and the rates of enzymatic repair of the adducts or their decay products. The principal modes of repair of methylating and ethylating agents are by glycosylase-catalysed depurination of 7-alkylguanine and 3-alkyladenine and by the dealkylation of O6-alkylguanine. The latter repair is accomplished by the transfer of the alkyl group to cysteine residues of acceptor proteins in a stoichiometric reaction. Repair by dealkylation cannot be detected by the standard methods used to measure DNA repair, but it is easy to estimate the acceptor activity in cell extracts by measuring the transfer of radioactive O6-alkyl groups in an exogenous DNA to protein. In extracts of cells treated with alkylating agents, the activity is depressed because the endogenous DNA is rapidly dealkylated, using up the acceptor activity. In many cell types, the decrease in activity is followed by an increase to the normal constitutive level. In other cells, there is no such adaptive response. We may catalogue the cell strains and lines investigated into three classes: (1) high constitutive activity (30 000-100 000 acceptor sites per cell) and rapid adaptive response (several hours), (2) high constitutive activity and very slow adaptive response, and (3) low constitutive activity. The cytotoxicity of methylating agents is highest for the last and lowest for the first class. Differences in constitutive levels of methyl accepting activity in extracts of human lymphocytes and in the acceptor activity in lung macrophages from smokers (low activity) and nonsmokers (high activity) have been observed.

Topics: Alkylation; Animals; Base Sequence; Cells, Cultured; DNA Repair; Guanine; Humans; Methylnitronitrosoguanidine; Nitroso Compounds

N-Ethyl-N-nitrosourea-induced mutations occurring within a 180-base pair target in the lacI gene of Escherichia coli were characterized by DNA sequencing. In total, 109 mutations were characterized in a wild-type background and 100 in an excision-repair-deficient (UvrB-) background. The majority of mutations induced in the two backgrounds (77 and 85%, respectively) were G:C = greater than A:T transitions, presumably resulting from miscoding O6-ethylguanine lesions. A significant proportion of the mutations (17 and 15%, respectively) were A:T = greater than G:C transitions, which probably result from miscoding O4-ethylthymine lesions. An analysis of the distribution of both types of mutation in the two backgrounds reveals two distinct influences of neighboring base sequence. These effects apply equally to both the G:C = greater than A:T and A:T = greater than G:C transitions. Firstly, miscoding lesions are most likely to occur at 5'-purine-G-3' or 5'-purine-T-3' sites. Secondly, the excision-repair machinery is less efficient at removing both O6-ethylguanine and O4-ethylthymine lesions which are flanked on both sides by G:C base pairs. Thus, in the wild-type spectrum an overabundance of transitions occurs at a 5'-G-G-G/C-3' or 5'-G-T-G/C-3' sequence (where the mutated base is underlined).

Topics: Base Sequence; DNA Repair; Escherichia coli; Ethylnitrosourea; Guanine; Methylnitronitrosoguanidine; Mutation; Thymine

DNA-adduct formation and induction of gene mutations were determined simultaneously after treatment with the four ethylating agents, ethyl methanesulfonate (EMS), ethylnitrosourea (ENU), diethyl sulfate (DES), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Both, in E. coli K-12 (NAL-resistance) and in V79 Chinese hamster cells in culture (HPRT-deficiency), the frequencies of mutation induction by all chemicals were the same when plotted against the amount of O6-ethylguanine formed in DNA, suggesting that this DNA adduct can be used as a common dosimeter for the comparisons of the frequencies of gene mutations induced by ethylating agents in various mutagenicity assay systems. Using ENU, such a comparison was performed between mutation induction in V79 cells in vitro and in the specific-locus assay in the mouse. The data indicate that at equal levels of O6-ethylguanine in the DNA of V79 cells and in testicular DNA from male mice treated with ENU, the frequencies of induced mutants in both assay systems were quite similar. These results support the concept that the determination of premutagenic DNA adducts in vivo can be used to monitor exposure to chemical mutagens and that genetic risk estimations may ultimately be performed on the basis of such measurements and of comparative mutagenesis in vitro and in vivo.

Topics: Alkylating Agents; Animals; Cell Line; Cells, Cultured; Cricetinae; Cricetulus; DNA; Environmental Exposure; Escherichia coli; Ethyl Methanesulfonate; Ethylnitrosourea; Guanine; Lung; Methylnitronitrosoguanidine; Mice; Mutation; Structure-Activity Relationship; Sulfuric Acid Esters