Metronidazole (Flagyl): lack of induction of sister-chromatid exchanges in CHO-K1 cells

328 X-linked recessive lethal mutations induced in late spermatids by hycanthone methanesulfonate were tested for coverage by duplications that comprised, in total, about 24% of the euchromatic X chromosome; 78 lethals appeared to be covered. Crossover localization tests of a random sample of 38 non-covered lethals revealed 4 chromosomes carrying a lethal within a duplicated segment. Lethals localized to a particular region were crossed to reference deficiencies and single-locus mutations, and inter se, to ascertain their genetic extent. The proportion of multi-locus deletions among these 78 covered and 4 non-covered lethals was 3/48, 1/10 and 13/24 for the distal, medial and proximal regions, respectively. A storage period of 9 days did not noticeably influence these proportions. In the sample of 38 non-covered lethals, and among 17 of the covered single-site lethals, 4 cases of strong crossover suppression were detected. Comparison of these results with data obtained with other mutagens suggests that induction of multi-locus deletions, and possibly of other types of chromosome rearrangement, could in part depend on other mechanisms than those acting in the formation of translocations and chromosome loss. For the purpose of mutagen testing, these findings imply that, in Drosophila, results in the regular genetic tests for chromosome breakage events do not always accurately predict the capacity of a mutagen to induce multi-locus deletions. This is of importance since transmissible multi-locus deletions have been considered a significant source of genetic damage in man.     

Mutagen sensitivity of Drosophila melanogaster. I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut^s, has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: (A) it is extremely sensitive to killing by MMS; (B) it is more mutable by MMS than the parent wildtype strain; and (C) it appears to possess mutator gene activity.     

Response of Escherichia coli to ethyl methanesulfonate: Influence of growth phase and repair ability on survival and mutagenesis

The effects of altering the cell growth rate (physiological state) and DNA repair capacity (genetic state) on susceptibility to inactivation and mutagenesis by ethyl methanesulfonate (EMS) were studied in 4 strains of E. coli. Logarithmic and stationary phase cells of the polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇ recA, and the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃ rec⁺, were exposed to EMS and the surviving fraction and mutant frequency determined. At the same EMS concentration both mutants were more susceptible to inactivation than the parental strains. In all 4 strains, log phase cells were more sensitive to inactivation than stationary cells. The surviving fraction of stationary cells exceeded log cells by a factor of 18 for polA, 6 for recA, and about 2 for the parental strains. In all strains, except recA, log phase cells exhibited higher spontaneous mutant frequencies than stationary phase cells. At the same concentration of EMS, survivors of both polA and recA showed more than 10-fold higher induced frequencies than the wild types. However, at the same survival levels the repair deficient mutants exhibited induced mutant frequencies comparable to the repair proficient strains. There was no significant effect of growth phase on EMS induced mutability in recA or the parental strains. In marked contrast, the polymerase I deficient mutant shows both a higher spontaneous frequency and a greater than 10-fold higher EMS induced mutant frequency in log phase cultures compared to stationary phase cultures. Our results support the hypothesis that cellular susceptibility to alkylating agents is influenced by both the genetic capability for repair and the particular physiological state of the cell.     

Repair of 3-methyladenine and 7-methylguanine in nuclear DNA of Chlamydomonas: Requirement for protein synthesis

The removal of 3-methyladenine and 7-methylguanine from nuclear DNA was determined following exposure of Chlamydomonas reinhardi to methyl methanesulfonate (MMS). The amount of 3-methyladenine in DNA was determined using an extract from Micrococcus luteus that has a 3-methyladenine-DNA glycosylase. The amount of 7-methylguanine was estimated by heating the DNA for 30 min at 70° followed by alkaline hydrolysis of the resulting apurinic sites. The molecular weight of the DNA was determined using alkaline sucrose gradients. The 3-methyladenine is removed with a half-life of 2–3 h whereas the 7-methylaguanine is removed with a half-life of 10–12 h. The rate of removal of the 7-methylguanine is more than an order of magnitude faster than the estimated non-enzymatic hydrolysis rate indicating the probability of enzymatic repair. Addition of cycloheximide immediately after MMS treatment inhibits the removal of 3-methyladenine and 7-methylguanine from DNA. If cycloheximide is added 1.5 h after treatment with MMS, there is much less inhibition of the removal of 3-methyladenine. These results are interpreted to mean that MMS induces the synthesis of 1 or more proteins that are required for the repair of 3-methyladenine from Chlamydomonas DNA.     

A protective effect of caffeine on drosophila larvae treated with methyl methanesulfonate.

Lethality induced in larval populations of Drosophila melanogaster was recorded after treatment with (1) caffeine, (2) MMS or (3) caffeine plus MMS. The mixture of caffeine plus MMS was less toxic than expected from the effects observed after treatment with either substance individually. It is postulated that in the combined treatment the caffeine, by inhibiting semiconservative DNA replication, allows for some additional time for repair of alkylated DNA by a repair pathway which is not sensitive to caffeine, possibly excision repair.     

Caffeine toxicity in drosophila strains having different MMS sensitivities.

Larvae of Drosophila melanogaster were fed with caffeine, and the induced lethality was recorded using two different methods. With a wild-type strain a whole dose-response curve was obtained. The two sexes were equally sensitive. Four genetically different strains, showing different MMS sensitivities, were tested with two caffeine doses. The strains differed significantly in their caffeine sensitivity, but there existed no correlation between MMS and caffeine sensitivity.     

Estimation of the effects of chemical mutagens: Lessons from radiation genetics

Years of work with ionizing radiations have given us a wealth of data on radiation-induced mutations. These data, which have given insights regarding the mutational processes, should form the background for all mutagenesis work. In chemical mutagenesis, as in radiation mutagenesis, it is important to know the shape of the dose-effect curve in order to make further interpretations and calculations. It is also important to be on the constant alert for new relations that can be explored.     

Detection of antigenically active mutant HGPRT after mutagenesis with Simian virus 40.

BCNU and 10 related chloroethylnitrosoureas were tested for their ability to induce sex-linked recessive lethals in Drosophila spermatozoa. All chloroethyl-nitrosoureas tested were potent mutagens.Among the substances with one chloroethylnitrosourea group, chlorozotocin, BCNU and methanesulfonyloxyethyl chloroethylnitrosourea exhibited the strongest mutagenic effects. Two hydroxyalkyl chloroethylnitrosoureas behaved as potent mutagens too, although the mutation frequencies obtained were one order of magnitude lower relative to the other substances.Among the compounds with two chloroethylnitrosourea groups, bisCNU-ethane and bisCNU-diphenylmethane were most active. When the interconnecting polymethylene chain was elongated from 2 methylene groups (bisCNU-ethane) to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. The mutagenic activity of polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length was not different from bisCNU-hexane.Differences in mutagenic activity were supposed to reflect different concentrations reaching the target cells, possibly in part as a result of differences in transportability of the substances.     

Comparative studies of the effects of carcinogenic and antitumor agents on the DNA replication of cultured mammalian cells

Cultured mouse L₅₁₇8Y cells were exposed to several carcinogenic and antitumor agents. After exposure to one of the agents, the cells were label with [³H]-thymidine for 20 min, and the DNA was subjected to alkaline sucrose gradient centrifugation immediately or after a chase period. This led us to classify the agents into 3 groups: (1) UV, 4-nitroquinoline-1-oxide (4NQO), N-methyl-N′-nitrosoguanidine (MNNG), nitrogen mustard and Mitomycin C. These were characterized by 20-min DNA labeling patterns showing the formation of small DNA and by the slowing down of their subsequent elongation. Replicated DNA strands would have gaps where “damage” was present on the parental strands. Subsequently, gap-filling replication would occur with or without repairing damage. (2) γ-rays. The 20-min DNA labeling profile displayed a larger size of DNA pieces and the subsequent elongation of this DNA was slightly affected. This probably due to a preferential depression of initiation DNA replication. (3) Methyl methanesulfonate (MMS) and low temperature (28°). The 20-min DNA labeling patterns were qualitatively similar to, but quantitatively different from those of non-irradiated control. The rate of DNA elongation was slightly retarded.     

The effects of radiation exposure-rate and exposure fractionation on the frequencies of recessive and dominant lethals in immature oocytes of Drosophila melanogaster

A measure of seedling viability was used to estimate the homozygous geneticc load of seedlings from mutagenized populations of Oenothera. The breeding protocol forced genomes to homozygosity. Pollen from control and mutagenized Oenothera hookeri T. and G. strain Johansen, a seven-paired lethal-free stock, was used to pollinate a translocation stock with balanced lethals. The hybrid formed a complete translocation ring and upon selfing yielded two types of plants, a translocation heterozygote similar to its hybrid parent and a seven-paired plant homozygous for the 7 chromosomes obtained from Johansen. Genetic markers allowed the identification of seven-paired seedlings in the cotyledon stage. Control hybrids averaged a recovery of 57.5% seven-paired seedlings. Hybrids obtained from plants that had been mutagenized by seed treatment with 15000 R X-rays, 0.04 M ethyl methanesulfonate (EMS), and 0.08 M EMS averaged 48.3%, 19.2%, and 6.0% recovery of seven-paired forms, respectively. The data are used to estimate the genetic load and lethal equivalents in each population. The implications of these results are evaluated with reference to mutation breeding of plant populations.     

Repair of UV-induced damage in wild-type and mutant strains of Schizophyllum commune

The basidiomycete fungus Schizophyllum commune was found to have both photo-repair and dark-repair systems for UV-induced damage. Three UV-sensitive mutants were isolated and characterized for ability to repair UV-induced damage in light and dark, and for cross-sensitivity to caffeine and methyl methanesulfonate. Two of the mutants were damaged, to different extents, in their capacity for excision repair; one of these mutants was also probably damaged in post-replication repair. The third mutant was damaged only in post-replication repair.     

Isolation and characterization of temperature-sensitive mutants in a Chinese hamster cell line

A replica plating method was used for the isolation of temperature-sensitive (ts) mutants after treatment of Chinese hamster cells with ethyl methanesulfonate (EMS). No significant increase in ts mutants was found after this treatment. The limitations and advantages of the replicating procedure to detect such differences, as well as an alternative method, are discussed.Mutants isolated were classified into two general groups—density-dependent and clear-cut—as measured by survival at low and high cell densities at the restrictive temperature. The density-dependent mutants may be truly “leaky”, losing a metabolite to the medium at an excessive rate at the restrictive temperature. On the other hand, the one clear-cut mutant analyzed extensively dies at a rate determined by its ability to utilize one or more components from the medium. It shows an inverse density relationship in rate of death, as inferred from rates of macromolecular synthesis, as opposed to its growth rate at the permissive temperature.     

Mutagenesis and transformation of C3H 10T12 cells treated with ethyl methanesulfonate

Survival, mutagenesis and transformation were measured in mouse embryo C3H $\text{10}\text{T}\text{1}\text{2}$ cells following treatment with ethyl methanesulfonate (EMS). Ouabain-resistant cells and transformed cells were isolated, and reconstruction experiments were carried out to determine the optimum conditions for the measurement of mutation and transformation frequencies. Survival was measured by plating efficiency; mutagenesis was measured in terms of the induction of cells able to form colonies in the presence of ouabain; and transformation was measured by the induction of cells forming either morphologically altered colonies on a monolayer of contact-inhibited cells or of cells capable of forming colonies in semi-solid media. When confluent monolayers were incubated for 4 h after treatment with EMS, to allow excision repair before the resumption of DNA synthesis, survival as well as the frequencies of both mutation and transformation increased. When this repair (or holding) period was extended to 24 h, the frequencies of mutation and transformation both decreased as compared to the 4-h holding period. Thus, the holding periods affect the frequencies of EMS-induced mutagenesis and transformation similarly.     

A yeast strain for simultaneous detection of induced mitotic crossing over,mitotic gene conversion and reverse mutation

A diploid yeast strain is described which can be used to study induction of mitotic crossing over, mitotic gene conversion and reverse mutation.Mitotic crossing over can be detected visually as pink and red twin sectored colonies which are due to the formation of homozygous cells of the genotype ade240/ade240 (deep red) and ade-2-119/ade2-119 (pink) from the originally heteroallelic condition ade2-40/ade2-119 which forms white colonies.Mitotic gene conversion is monitored by the appearance of tryptophan non-requiring colonies on selective media. The alleles involved are tryp5-12 and trp5-27 derived from the widely used strain D4.Mutation induction can be followed by the appearance of isoleucine non-requiring colonies on selective media. D7 is homoallelic ilv1-92/ilv1-92. The isoleucine requirement caused by ilv1-92 can be alleviated by true reverse mutation and allele non-specific suppressor mutation.The effects of ethyl methanesulfonate (EMS), nitrous acid, ultraviolet light and hycanthone methanesulfonate were studied with D7 stationary phase cells. Mitotic crossing over as monitored by red/pink twin sectored colonies was almost equally frequent among normal and convertant cells. This showed again that mitotic recombination is not due to the presence fo a few cells committed to meiosis in an otherwise mitotic cell population.The dose-response curves for induction of mitotic gene conversion and reversion of the isoleucine requirement were exponential. In contrast to this, the dose-response curve for induction of twin sectored red and pink colonies reached a plateau at doses giving about 30% cell killing. This could partly be due to lethal segregation in the progeny of treated cells.None of the agents tested would induce only one type of mitotic recombination, gene conversion or crossing over. There was, however, some mutagen specificity in the induction of isoleucine prototrophs.     

Mutagenesis at the ad-3A and ad-3B loci haploid UV-sensitive strains of Neurospora crassa. III. Comparison of dose-response curves for inactivation and mutation induced by gamma-rays

Gamma-Ray-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 6 different UV-sensitive strains and a standard wild-type strain. The 6 strains show varying degrees of sensitivity to gamma-ray-induced inactivation, with the relative sensitivity at 37% survival being uvs-6 greater than upr-1 greater than uvs-2 greater than uvs-3 greater than wild-type greater than uvs-5 greater than uvs-4. Studies on the induction of ad-3 mutants by gamma-rays show that when the dose-response curve (expressed in terms of ad-3 mutants among the surviving colonies) of the UV-sensitive strains are compared with wild-type, the 2 excision-repair-deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, uvs-3 exhibits reduced ad-3 mutant frequencies whereas both uvs-4 and uvs-5 show lower mutant frequencies than wild-type.