Effect of N-nitroso-N-methylurea on viability and mutagenic response of repair-deficient strains of Escherichia coli

Various E. coli mutants, deficient in DNA repair, differed in their response to increasing concentrations of N-nitroso-N-methylurea (NMU).Loss of viability due to exposure to NMU was greatest in those strains with a reduced capacity for repair of single-strand breaks. Viability of wild-type and uvrA^? strains was not affected by NMU concentrations up to 3.0 mM. Some loss of viability occurred, at the higher NMU concentrations, in both strains carrying exrA^? while strains carrying uvrA^?polA^? or recA^? were the most sensitive. The results support the hypothesis that the lethal effect of NMU on repair-deficient E. coli was due to its ability to induce single-strand breaks.Induction of mutations by NMU was observed in all the strains used and the results suggested that NMU damage per se was the major mutational event. The dose response curve for induction of revertants by NMU was, however, influenced by the repair system(s) present. The number of revertants scored at the higher NMU concentrations was greater in those strains lacking the recA and polA dependent repair functions than in the wild-type strain. However, at NMU concentrations below 2.0 mM the numbers of revertants induced in exrA^? carrying strains, prossessing accurate rec-dependent repair, were lower than the comparable wild-type values. The evidence suggests that the uvrA gene product also acts on some, possibly non-mutagenic, types of NMU damage and that error-prone repair of these lesions increases the number of potential revertants.     

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

DNA strand breakage caused by dichlorvos, methyl methanesulphonate and iodoacetamide in Escherichia coli and cultured Chinese hamster cells

The DNA damaging properties of dichlorvos (2,2 dichlorovinyl dimethyl phosphate), methyl methanesulphonate (MMS) and iodoacetamide (IAA) have been studied, using alkaline sucrose sedimentation. In a strain of E. coli deficient in DNA polymerase I (polA) both dichlorvos and MMS caused random strand breakage, MMS being about twice as efficient as dichlorvos on a molar basis. In pol⁺ bacteria, DNA strand breaks or alkali labile bonds were detected following treatment with roughly five-fold higher concentrations of MMS but at similar high concentrations of dichlorvos there was an all or none breakdown of DNA molecules to fragments of very low molecular weight which correlated well with lethality.DNA synthesized after treatment of pol⁺ and polA bacteria with MMS was of low molecular weight, indicating the presence of discontinuities. With dichlorvos, the effect was much smaller.Apparent all-or-none DNA breakdown was also found when the polA strain of E. coli was treated with low concentrations of iodoacetamide, an agent that does not detectably alkylate DNA. At higher concentrations the breakdown was suppressed and random strand breakage occurred instea. These effects did not occurr with pol⁺ bacteria and correlated well with the greater sensitivity to iodoacetamide of the polA strain in survival experiments. We suggest that the major DNA damage resulting from treatment with iodoacetamide and dichlorvos arises indirectly through alkylation of other cellular constituents and consequent uncontrolled nuclease attack on the DNA. Discontinuities in newly synthesized DNA and mutagenesis following dichlorvos treatment, however, presumably result from direct alkylation of DNA.Strand breakage caused by dichlorvos and MMS in Chinese hamster cells tended to correlate with the extent to which these agents alkylate DNA, but survivval tended to correlate with the alkylation of protein.     

Mutagenicity of dichlorvos and methyl methanesulphonate for Escherichia coli WP2 and some derivatives deficient in DNA repair

The mutagenic and lethal action of methyl methanesulphonate (MMS) and dichlorvos (DDVP) has been studied on Escherichia coli WP2 and some derivatives deficient in DNA repair genes. The exrA⁺ and recA⁺ alleles were necessary for significant mutagenesis by either compound, and the uvrA gene affected neither the lethal nor mutagenic responses. Increased sensitivity to both compounds was shown by the exrA and uvrAexrA strains and in a more pronounced way by the uvrApolA, recA, and uvrAexrApolA strains.Bacteria deficient at the polA locus were 2 and 3 times more mutable by DDVP and MMS respectively, consistent with the hypothesis that the absence of the polA system for the repair of single-strand gaps results in a greater proportion of the total repair being channelled through the error-prone exrA⁺/recA⁺-dependent system. Single-strand breaks were detectable by alkaline sucrose gradient centrifugation after both MMS and DDVP treatment of polA bacteria. Thus in all the tests carried out, both compounds showed similar patterns of activity, and the results are consistent with their known ability to alkylate DNA. The chief differences were quantitative; sensitivity increases were far more pronounced with MMS which was also a far more potent mutagen than DDVP.     

Heritable translocation test and dominant-lethal assay in mice with methyl methanesulfonate.

A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F1 males varied between 2 and 99%. Transmission of partial sterility and translocations was confirmed in the F2 generation. There were no partially sterile or sterile males among the 245 controls.     

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.     

Reversions of the two missense and one nonsense trp-mutations induced by UV-light or methyl methanesulfonate in E. coli wild type and its polAi and uvrE502 mutants.

Two missense mutations, trpA58 and trpA78, and one nonsense mutation-trp-ochre, were used to determine the types of base-pair substitution caused by ultra, violet irradiation and methyl methanesulfonate (MMS) in Escherichia coli. UV irradiation of the wild-type bacteria led to the formation of revertants mainly arising as a result of GC yields AT transitions (suppressor revertants of the trpA58 mutant). True revertants of the trp- mutant (arising via transitions of AT pairs) and 5-methyl tryptophan-sensitive (MT-s) Trp+ of the trpA78 mutant (arising via unidentified transversions) occurred at a lower frequency. The polAI mutation did not change the frequency of the UV-induced transitions GC yields AT or that of the substitutions of the AT pairs. The uvrE502 mutation significantly increased the frequency of the UV-induced revertants arising via the transition GC yields AT. Treatment of the wild-type bacteria with MMS resulted in the formation of revertants mainly due to the GC yields AT substitution, and with a lower frequency to the AT yields GC transitions. MMS also induced, with a low frequency, some transversions. The frequency of the MMS-induced GC yields AT transitions was enhanced in the uvrE502 mutant. On the other hand, the uvrE502 mutation eliminated or significantly lowered MMS-induced revertants arising as a result of AT yields GC transitions or transversions.     

Mutagenesis by nitrosoguanidine, ethyl methanesulfonate, and mutator gene mutH in continuous cultures of Escherichia coli.

Induction of T5-R mutations by alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS) was examined in glucose limited chemostat cultures of non-mutator and mutator (mutH) bacteria. In agreement with the proposal that NTG mutagenizes DNA at the replication fork, this mutagen (6.8 X 10-minus 6 M) showed replication-dependent mutagenesis in continuous culture. EMS (5-10-minus M)) induced mutagenesis could not be correlated with growth rate, which probably means that induction of mutagenic lesions (promutations) by this mutagen does not involve replicating genes. A large synergic response was found for the mutH gene in combination with NTG, supporting the hypothesis that the mutH gene product acts during DNA replication.     

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.     

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.     

A comparison of mutation induction by 14 MeV fast neutrons and 137 Cs -rays in meiotic spermatocytes in the silkworm

Barley seeds were treated with methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), stored at 15% water content and washed for 16–24 h. These treatments resulted in an increase of toxic and genetic effects. In teh DNA of embryos of such stored MMS- and EMS-treated seeds, a strong enhancement of the amount of single-strand breaks and/or alkali-labile sites took place. In contrast, the amount of alkylated sites, particularly of 7-methylguanine, was somewhat lower. It can be that the depurination and/or backbone breakage, which proceeds during the storage period, is responsible for the enhancement of toxic and genetic effects, whereas the influence of the alkylation of DNA during the storage period by the unreacted residual mutagen is negligible.     

Dose-rate effects of ethyl methanesulfonate on survival and mutation induction in cultured Chinese hamster cells

The dose-rate effects of ethyl methanesulfonate (EMS) on the survival and induction of mutations in Chinese hamster Don cells were investigated. The most effective time of exposure to EMS for reducing the surviving fraction of cells was 4 h, shorter and longer exposure times being less effective. The threshold or minimal concentration of EMS giving a surviving fraction of 0.5 was 0.05 mg/ml. The minimal effective time of exposure to EMS for cell death was 1 h. Corrected survival curves showed that longer exposure times at lower dose rates of EMS had less cytotoxic effect than shorter exposure times at higher dose rates.After exposure of Don cells to various doses of EMS for various times, the frequencies of mutations resistant to 6-thioguanine (6TG) were measured. An exposure time of 4 h produced a lower mutation frequency than shorter or longer exposure times that resulted in the same surviving fraction of cells. An exposure time of 20 h produced the highest induced mutation frequency.This system using cultured Chinese hamster cells should be useful as a sensitive procedure for detecting the mutagenic actions of chemicals.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae V. Ethyl methanesulfonate

Mitochondrial genomes are more sensitive to the lethal action of EMS than are nuclear genomes of S. cerevisiae. EMS induces efficiently only some types of mutation in nuclear genomes of yeast, and probably the same is true for induction of mutations non-lethal to the mitochondrial genomes.     

Inactivation and mutation of coliphage T4 by aliphatic nitrosamides and methanesulphonates: in vitro recovery of infectivity of T4 inactivated by isopropyl methanesulphonate.

The inactivation and mutation (to r phenotype) of extracellular coliphage T4 wild-type by the monofunctional alkylating agents N-methyl- and N-ethyl-N-nitrosourea and isopropyl methanesulphonate were investigated. The rate and extent of change in phage infectivity observed during the post-treatment period were found to correlate with what is known of the mechanisms by which these agents react in vitro. Loss of phage infectivity was found to occur during the period following treatment with these agents, but that resulting from treatment with isopropyl methanesulphonate was preceded, in the first 24 to 48 h, by a recovery of infectivity. This suggested that changes in phage infectivity occurring after treatment with monofunctional alkylating agents are resultant of various processes which diversely promote loss and recovery of infectivity. The mutagenicity of N-methyl-N-nitrosourea was similar to that of its ethyl homologue at a level of phage survival of 4 x 10-3, but less than that of isopropyl methanesulphonate. At a level of survival of 3 x 10-2 ethyl methanesulphonate was a mutagenic as its isopropyl homologue, but methyl methanesulphonate was only slightly if at all mutagenic. These results could not be correlated with the compounds' reaction mechanisms. The efficiency of isopropyl methanesulphonate (compared with its toxicity to phage) was found to decrease as the severity of the dose was increased.     

Comparison of radiation-and chemically-induced dominant lethal mutations in male mice

Ionizing radiation-induced dominant lethal mutations in all spermatogenic stages. After irradiation of male mice with 200 R the yield of induced mutations in early spermatids was twice the yield in spermatozoa, late spermatids, and spermatocytes. After irradiation with 400 R or 800 R the spermatocytes were the most sensitive stage for the induction of dominant lethal mutations. The frequency of radiation-induced dominant lethal mutations in postspermatogonial stages was dose-dependent. The yield of dominant lethal mutations in spermatogonia was independent of the dose.