enng and diethyl-sulfate

Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.

Topics: Alkylating Agents; Animals; Epichlorohydrin; Ethyl Methanesulfonate; Ethylene Dibromide; Ethylnitrosourea; Evaluation Studies as Topic; Male; Methylnitronitrosoguanidine; Mice; Micronucleus Tests; Reticulocytes; Sulfuric Acid Esters

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

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (ssb) or alkali-labile sites were measured by centrifugation in alkaline sucrose gradients after lysis in alkali. 4 agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate (EMS) and diethyl sulphonate (DES). The compounds differed greatly in their potency to induce the lesions measured when compared on a molar basis, but comparison at equicytotoxic doses showed relatively small differences. Upon prolonged incubation of the DNA in alkali, the number of ssb increased considerably. DNA from untreated cells showed biphasic kinetics: slow ssb formation for about 10 h, then the rate increased and remained constant for up to 40 h. Treated cells showed an accelerated, dose-dependent linear generation of ssb for 10 h, followed by a short plateau; then ssb were formed again at a constant rate, somewhat higher than that in controls. Ssb formed in the initial phase are ascribed to phosphotriester hydrolysis, those after the plateau to unidentified causes. Zero intercepts appeared to be a measure of apurinic sites generated intracellularly. A 24-h repair period preceding lysis reduced the ENNG intercept, but not that of DES. Rapid degradation of DES during the 1-h treatment occurred, so most "apurinic-site lesions" were induced in the beginning of exposure and possibly were already repaired at the end. The types of lesion distinguished (reparable and non-reparable apurinic sites, phosphotriesters) appeared of little consequence for cell survival.

Topics: Alkylating Agents; Animals; Cell Line; Cell Survival; Cricetinae; Cricetulus; DNA; DNA Repair; Dose-Response Relationship, Drug; Ethyl Methanesulfonate; Ethylnitrosourea; Female; Hydrogen-Ion Concentration; Methylnitronitrosoguanidine; Ovary; Sulfuric Acid Esters; Time Factors