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.     

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.     

Choloroquine inhibition of repair of DNA damage induced in mammalian cells by methyl methanesulfonate

Chloroquine (ClQ) inhibited the repair of DNA damage produced in cultured rat liver cells by methyl methanesulfonate (MMS). MMS caused fragmentation of single-strand DNA in alkaline sucrose gradients. Repair of the damage was followed by observing the restoration of the normal sedimentation pattern at intervals after treatment. Repair was significant by 7 h and nearly complete at 24 h. Addition of ClQ during the repair peiod markedly reduced the rate of repair. Also, ClQ increased the lethality of MMS, which could be due to the inhibition of repair. ClQ was found to inhibit protein synthesis, but the effect on repair is probably not due entirely to this action since comparable inhibition of protein synthesis by cycloheximide produced a lesser degree of delay in repair.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

The recovery of mammalian cells treated with methyl methanesulfonate, nitrogen mustard or UV light. I. The effect of alkylation products on DNA replication.

CHO cells were synchronized in G1 phase and treated with MMS or HN2. The subsequent rate of DNA replication was found to be reduced in a dose-dependent manner. In addition, 2 X 10(-3 M and 3 X 10(-3) M MMS resulted in a 3--4 h delay prior to the initiation of S phase. If the cells were held for 8 h in hydroxyurea after MMS treatment, no subsequent lag in DNA synthesis was seen after removal of the hydroxyurea. The entry of confluent cells into S phase was found to be delayed 7 h upon trypsinizing and replating. Treatment of these cells with MMS resulted in a reduced rate of DNA replication, but no further delay in its initiation. Repair replication was found to continue at a constant rate for at least 12 h following MMS treatment of cells under all of these conditions. At the concentrations used in these experiments MMS severely inhibited the rate of protein synthesis, but HN2 had little effect. By comparing both the kinetics of repair replication and recovery of protein synthesis with the rate of DNA replication, it was concluded that the initial, severe reduction in rate following MMS treatment was probably due to an inhibition of protein synthesis.     

Genetic analysis of purple adenine (ad-3) mutants induced by methyl methanesulfonate in Neurospora crassa

The mutagenicity of methyl methanesulfonate (MMS) was studied in a genetically marked two-component heterokaryon of Neurospora crassa. Types of genetic alterations detectable in this system are (I) point mutations in the ad-3A and ad-3B loci; (2) multilocus (chromosome) deletions in the ad-3 region, and (3) recessive lethal mutations in the whole genome. Study of the inactivation kinetics of the heterokaryotic and homokaryotic conidial fractions has made it possible to distinguish between nuclear and cytoplasmic inactivation.Forward mutations in the ad-3 region induced by MMS in the heterokaryotic fraction of conidia were obtained by a direct method with the following results: (I) The overall ad-3 forward mutation frequency increases in proportion to the 1.91 power of the concentration of MMS. (2) The forward mutation frequency of point mutations at the ad-3A and ad-3B loci increases in proportion to the 1.68 power of the concentration. (3) The forward mutation frequency of chromosome deletions in the ad-3 region increases more than exponentially with increasing concentrations of MMS. (4) After treatment for 300 min with 20 mM MMS, 15.5% of the ad-3 mutations are multilocus deletions. Tests for genotype and allelic complementation of the point mutations showed that (I) the ratio between ad-3B and ad-3A mutants was 1.75, (2) 52.1% of the ad-3B mutants showed allelic complementation, with 39.2% non-polarized and 12.9% polarized complementation patterns and 47.9% noncomplementing mutants, and (3) both the ratio between point mutations in the ad-3A and ad-3B loci and the spectrum of complementation patterns among the ad-3B mutants were independent of MMS concentration.     

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.     

Reassociation of human lymphoblastoid cell DNA repair replicated following methyl methanesulfonate treatment

The reassociation rates of repair replicated DNA of two human lymphoblastoid cell lines, the WIL₂-A3 ‘normal’ line and the RAJI line of Burkitt's lymphoma, were examined using the DNA/DNA ‘C₀t’ hybridization technique. The cells were treated with methyl methanesulfonate (MMS), an alkylating agent and mutagen, to induce the repair.The incorporated repair replication radioactivity in highly repetitive sequences of WIL₂-A3 cell DNA reassociates as expected for a randomly distributed incorporation. The reassociation of repair radioactivity in sequences of fewer numbers of copies, however, is less than expected for a random distribution. It is less than that occurring for semiconservatively synthesized DNA of WIL₂-A3 cells co-incubated with the repair labeled DNA as an internal control.The observed difference could be due to an over-representation of repair replication radioactivity in DNA sequences with fewer copies. It is unlikely to be due to residual alkali labile damage resulting from MMS treatment, since a similar difference was not observed when semiconservatively labeled DNA from cells which had been treated with MMS for the same time and at the same concentration as in the repair experiments was substituted for repair replicated DNA in the reassociation reactions. Other possible causes of the apparent difference in the reassociation rates observed are discussed.     

Chemical induction of streptomycin-resistant mutations in Escherichia coli. Dose and mutagenic effects of dichlorvos and methyl methanesulfonate

A procedure for the quantitative determination of induced streptomycin-resistant mutants in E. coli was applied to study and compare mutation induction by the organophosphate dichlorvos and by methyl methanesulfonate (MMS). Both compounds increased the frequency of mutants even under conditions where no inactivation of cell was observed. Mutation induction by these agents as a function of both concentration and exposure time was measured. The dose-response curves found with both mutagens were non-linear; atp higher doses more mutants were induced per unit dose than at lower doses. Possible relationships between dose-effect curves and the chemical nature of alkylating mutagenic agents are discussed.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures

Sodium selenite (Na₂Se0₃) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl aminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 × 10^-6 M Na₂SeO₃ resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na₂SeO₃ concentrations (7.90 × 10^?6 and 1.19 × 10^?5 M) resulted in a three-fold increase in the SCE frequency above background level (6–7 SCEs/cell). Exposure of lymphocytes to 1 × 10^?4 M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 ± 0.75 while a similar exposure to 2.7 × 10^?5 M N-OH-AAF resulted in 13.61 ± 0.43 SCEs/cell. Simultaneous addition of the high Na₂Se0₃ concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25–30% and 11–17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures.

Sodium selenite (Na2SeO3) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetylaminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 X 10(-6) M Na2SeO3 resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na2SeO3 concentrations (7.90 X 10(-6) and 1.19 X 10(-5) M) resulted in a three-fold increase in the SCE frequency above background level (6--7 SCEs/cell). Exposure of lymphocytes to 1 X 10(-4) M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 +/- 0.75 while a similar exposure to 2.7 X 10(-5) M N-OH-AAF resulted in 13.61 +/- 0.43 SCEs/cell. Simultaneous addition of the high Na2SeO3 concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25--30% and 11--17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

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.     

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.     

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.     

Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice

The molecular dosimetry of methyl methanesulfonate (MMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 100 mg/kg of [3H]MMS and methylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 3-week period. The methylations per sperm head paralleled the dominant lethal frequency curve for MMS, reaching a maximum of between 22 and 26 million methylations per vas sperm head 8-11 days after treatment. Methylation of sperm DNA was greatest at 4 h (the earliest time point studied) after treatment, with 16.6 methylations/10(5) deoxynucleotides. DNA methylation gradually decreased during the subsequent 3-week period. The methylation of germ-cell DNA did not increase in the stages most sensitive to MMS (late spermatids leads to early spermatozoa) and was not correlated with the dominant lethal frequency curve for MMS. However, methylation of protamine did increase in the germ-cell stages most sensitive to MMS, and showed an excellent correlation with the incidence of dominant lethals produced by MMS in the different germ-cell stages. The pattern of alkylation produced by MMS in the developing germ-cell stages of the mouse is similar to that found for EMS. However, for equimolar exposures, MMS alkylates the germ cells 5-7 times more than does EMS. Hydrolyzed samples of protamine from [3H]MMS-exposed animals were subjected to thin-layer chromatography and amino acid analysis. Both procedures showed that most of the labeled material recovered from the hydrolysates co-chromatographed with authentic standards of S-methyl-L-cysteine. The amino acid analyses showed an average of approximately 80% of the labeled material eluting with S-methyl-L-cysteine. The mechanism of action of both MMS and EMS on the developing germ cells appears to be similar. The occurrence of S-methyl-L-cysteine as the major reaction product in sperm protamine after MMS exposure supports our initial model of how dominant lethals are induced in mouse germ cells by these chemicals: Alkylation of cysteine sulfhydryl groups contained in mouse-sperm protamine blocks normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Subsequent stresses produced in the chromatin structure eventually lead to chromosome breakage, with resultant dominant lethality.     

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.     

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.     

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.     

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.     

Mass spectrometry-based quantification of the cellular response to methyl methanesulfonate treatment in human cells

Faithful transmission of genetic material is essential for cell viability and organism health. The occurrence of DNA damage, due to either spontaneous events or environmental agents, threatens the integrity of the genome. The consequences of these insults, if allowed to perpetuate and accumulate over time, are mutations that can lead to the development of diseases such as cancer. Alkylation is a relevant DNA lesion produced endogenously as well as by exogenous agents including certain chemotherapeutics. We sought to better understand the cellular response to this form of DNA damage using mass spectrometry-based proteomics. For this purpose, we performed sub-cellular fractionation to monitor the effect of methyl methanesulfonate (MMS) treatment on protein localization to chromatin. The levels of over 500 proteins were increased in the chromatin-enriched nuclear lysate including histone chaperones. Levels of ubiquitin and subunits of the proteasome were also increased within this fraction, suggesting that ubiquitin-mediated degradation by the proteasome has an important role in the chromatin response to MMS treatment. Finally, the levels of some proteins were decreased within the chromatin-enriched lysate including components of the nuclear pore complex. Our spatial proteomics data demonstrate that many proteins that influence chromatin organization are regulated in response to MMS treatment, presumably to open the DNA to allow access by other DNA damage response proteins. To gain further insight into the cellular response to MMS-induced DNA damage, we also performed phosphorylation enrichment on total cell lysates to identify proteins regulated via post-translational modification. Phosphoproteomic analysis demonstrated that many nuclear phosphorylation events were decreased in response to MMS treatment. This reflected changes in protein kinase and/or phosphatase activity in response to DNA damage rather than changes in total protein abundance. Using these two mass spectrometry-based approaches, we have identified a novel set of MMS-responsive proteins that will expand our understanding of DNA damage signaling.