Mutagenicity of derivatives of the flame retardant tris(2,3-dibromopropyl) phosphate: halogenated propanols.

The sensitivity of larval populations of Drosophila melanogaster to the lethal action of methyl methanesulfonate (MMS) was determined. Wild-type strains were compared with strains carrying X-linked mutations that increase mutagen sensitivity. The determination of dose—response relationships for MMS-induced lethality allowed for a quantitative comparison of the MMS sensitivity of the mutants. The sensitivity difference, measured by the LD-50 values, between the most resistant and the most sensitive stock used in this study was 40-fold. Stocks containing mutations in the meiotic genes mei-41 and mei-9 were by far the most sensitive ones. These mutants are known to be repair-deficient.The meiotic mutants were tested in various stocks with different genetic backgrounds. It turned out that the larval MMS sensitivity strongly depended on the genotype of the parental females used to obtain the larval populations for MMS treatment. These maternal effects were not simulated by an age-dependent variation in MMS sensitivity because no differences in developmental time between the strains with different genetic constitution were found. Furthermore, a maternal effect on the relative frequency of spontaneous lethality of genetically identical mutant progeny derived from different types of female was demonstrated.These maternal effects, both on spontaneous lethality and on larval MMS sensitivity, are of interest because they extend beyond the embryonic stages of development.     

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.     

Isolation and characterization of MMS-sensitive mutants of Neurospora crassa

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.     

Caffeine interactions with methyl methanesulphonate, hycanthone, benlate, and cadmium chloride in chromosomal meiotic segregation of Saccharomyces cerevisiae

Interactions of caffeine with chemicals known for their effects on chromosomal segregation during meiosis of Saccharomyces cerevisiae were studied. It appears that caffeine does interfere with the action of other compounds during the different phases of meiosis. Treatments with methyl methanesulphonate (MMS) and cadmium chloride (CdCl2) resulted in a synergistic effect consisting of an increase in the frequency of recombination. The greatest effects were found on the induction of diploid spores: MMS, hycanthone, and distamycin demonstrated strong, benlate little synergistic action. CdCl2 demonstrated antagonism to caffeine by counter-inhibiting its effect on the induction of diploids. Concerning disomic induction: caffeine reduced (or left unchanged) the effect on non-disjunction when MMS and hycanthone were used. Simple additive effects were caused in conjunction with distamycin, benlate, and (in small doses) CdCl2. 2 mg of caffeine/ml in treatments with CdCl2 resulted in a very high frequency of disomic clones.     

Genetic evidence that cocaine and caffeine stimulate locomotion in mice via different mechanisms

The effects of cocaine and caffeine on motor activity in two mouse strains 129/OlaHsd (129) and C57BL/6J (C57) were compared. The former mice exhibited lower basal motor activity than the latter. Cocaine (3, 10, 30 mg/kg) injected i.p. in habituated C57 mice produced a dose-dependent increase in rearing, motility and locomotion. In 129 mice, little or no stimulation was seen and only with the highest dose of cocaine. In both strains caffeine (3, 15, 30 mg/kg) produced a dose-dependent increase in rearing, motility and locomotion. The effect of caffeine on rearing was greater in C57 than in 129 mice, but motility and locomotion were stimulated approximately to the same degree in both strains. Thus, differences in the sensitivity to caffeine and cocaine between mouse strains provide genetic evidence that these two stimulants probably produce locomotor stimulation via somewhat different mechanisms.     

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.     

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.     

Cross sensitivity of mutator strains to physical and chemical mutagens.

Ten different mutator strains of Saccharomyces cerevisiae were tested for cross sensitivity to two alkylaitng agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), to determine if any of them are defective in the repair systems which normally deal with damage caused by these agents. For one of the mutators, namely mut2-1, it was shown by genetic analysis that mutator activity and MMS sensitivity are both controlled by the same gene. Two mutants, mut2-1 and mut7-1, were found to be sensitive to MMS but normal to ultraviolet and gamma-rays. Another group is represented by mut1, mut6 and mut8 which are not sensitive to any of the mutagens tested so far. Mutator strain mut2-1 was also shown not to be significantly altered for levels of UV-induced forward and reverse mutations. These observations lend support to the idea of multiple repair systems that deal with DNA damage caused by different agents and also show that mutator activity can often result from the loss of normal cellular repair systems.     

Phosphorylation of Mcm2 modulates Mcm2-7 activity and affects the cell's response to DNA damage

The S-phase kinase, DDK controls DNA replication through phosphorylation of the replicative helicase, Mcm2-7. We show that phosphorylation of Mcm2 at S164 and S170 is not essential for viability. However, the relevance of Mcm2 phosphorylation is demonstrated by the sensitivity of a strain containing alanine at these positions (mcm2(AA)) to methyl methanesulfonate (MMS) and caffeine. Consistent with a role for Mcm2 phosphorylation in response to DNA damage, the mcm2(AA) strain accumulates more RPA foci than wild type. An allele with the phosphomimetic mutations S164E and S170E (mcm2(EE)) suppresses the MMS and caffeine sensitivity caused by deficiencies in DDK function. In vitro, phosphorylation of Mcm2 or Mcm2(EE) reduces the helicase activity of Mcm2-7 while increasing DNA binding. The reduced helicase activity likely results from the increased DNA binding since relaxing DNA binding with salt restores helicase activity. The finding that the ATP site mutant mcm2(K549R) has higher DNA binding and less ATPase than mcm2(EE), but like mcm2(AA) results in drug sensitivity, supports a model whereby a specific range of Mcm2-7 activity is required in response to MMS and caffeine. We propose that phosphorylation of Mcm2 fine-tunes the activity of Mcm2-7, which in turn modulates DNA replication in response to DNA damage.     

Rates of micronuclei induction in different mouse strains

It has previously been shown that the inbred mouse strain MS/Ae was more sensitive in the micronucleus test to several mutagenic agents than outbred mice. To elucidate the possible influence of inbreeding, several inbred strains including MS/Ae, AKR, BALB/c, C57 BR were compared to the two OF1 and NMRI outbred strains. The 3 mutagenic agents MNNG, MMC and MMS all induced a significantly higher number of micronuclei in the MS/Ae strain than in any of the other mouse strains. AKR was especially resistant to the alkylating agents MMS and MNNG. Hence, except for the MS/Ae mouse strain, no inbred strain showed a systematically higher sensitivity than the outbred strains for all of the 3 mutagenic agents used.     

Chromosomal analysis in mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) and methyl and ethyl methanesulfonate (MMS and EMS)

Chromosome aberrations were analyzed at the first-cleavage metaphase of mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) as well as to methyl and ethyl methanesulfonate (MMS and EMS). The frequencies of chromosome aberrations markedly increased with dose of UV as well as with concentration of MMS and EMS. In the UV-irradiation group, the frequency of chromosome-type aberrations was much higher than that of chromatid-type aberrations. About 90% of chromosome aberrations observed in the eggs following MMS and EMS treatment to sperm were chromosome type in which the frequency of chromosome fragments was the highest. The effects of UV on the induction of chromosome aberrations were clearly potentiated by post-treatment incubation of fertilized eggs in the presence of Ara-C or caffeine, but the effects of MMS and EMS were not pronounced by post-treatment of Ara-C or caffeine. The results indicate a possibility that UV damage induced in mouse sperm DNA is reparable in the eggs during the period between the entry of sperm into the egg cytoplasm and the first-cleavage metaphase.     

Cytotoxicity of monofunctional alkylating agents methyl methanesulfonate and methyl-N′-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN₁ and SN₂ mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O⁶ position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10T1/2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10T1/2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10T1/2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10T1/2 cells are treated with MMS. Furthermore, MMS toxicity for 10T1/2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10T1/2 cells are exposed during the S phase and the effect of potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10T1/2 cells treated with MMS. MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMS kill 10T1/2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.     

The Isolation of Mms- and Histidine-Sensitive Mutants in NEUROSPORA CRASSA

A simple method of replica plating has been used to isolate mutants of Neurospora crassa that have increased sensitivity to methyl methanesulfonate (MMS) and/or to histidine. Twelve mutants with increased sensitivity to MMS and one mutant with increased sensitivity to histidine showed Mendelian segregation of the mutant phenotypes. Three mutants were mapped to loci not previously associated with MMS sensitivity. Two others were allelic to the UV- and MMS-sensitive mutant, mei-3. Survival curves indicate that conidia (mutant or wild-type) survive on much higher concentrations of MMS at 25° than at 37°. In contrast, mycelial growth is more resistant to MMS at 37°. The possibility of qualitatively different repair processes at these two temperatures is discussed.     

Isolation and characterization of second chromosome mutagen-sensitive mutations in Drosophila melanogaster

We have undertaken the study of a collection of 32 Drosophila melanogaster mus strains selected on the basis of developmental sensitivity to the DNA-damaging agents, methyl methanesulfonate (MMS), N-acetyl-2-aminofluorene (AAF), nitrogen mustard (HN2), and gamma-radiation. In total, 18 of these strains are sensitive to MMS. In turn, 14 of these exhibit unconditional MMS sensitivity (one of the latter mutants is lethal at 29 degrees C), whereas the other 4 are sensitive to MMS only at higher temperatures. Detailed analysis of the 7 strongest MMS-sensitive strains reveals that they identify 4 new second chromosome mus loci. Two mus loci are each represented by two alleles. One mutant (mus205B1) is allelic to a previously characterized mus locus. Different MMS-sensitive mutants display patterns of mutagen cross-sensitivity (to AAF, HN2, benzo[a]pyrene (BP), and gamma-rays) that parallel the range of responses seen in previously recovered X-linked and autosomal mus loci. In general, mutations that are strongly sensitive to MMS are also sensitive to one or both of the procarcinogens, AAF and BP, as opposed to HN2 and gamma-radiation. In contrast, the moderately MMS-sensitive mutations are sensitive to HN2 and gamma-rays, but not to AAF or BP. Of the 14 mus strains that are not sensitive to MMS, 5 are sensitive to AAF, another 5 are sensitive to HN2, and the remaining 4 are sensitive to gamma-rays.     

The partial recA-lexA dependence of the adaptive response of Escherichia coli to exposure to methylmethane sulfonate

A study was made of the adaptive response to methylmethane sulfonate (MMS) in E. coli. (18 strains of B, WP2, and H/r30 groups, including three strains of bacteria with pKM101 plasmid). The adaptation of wild type cells and uvrA- and uvrB- mutants to non-lethal concentrations of MMS (10-30 mkg/ml during 90-120 min) leads to a significant increase in their resistance to lethal MMS concentrations (10-30 mM for 10-120 min): the dose modifying factor (DMF) being 1.5-1.8. In single recA or lexA mutants (or double recA uvr- and lexA uvr- mutants) the efficiency of adaptive response to MMS was significantly lower: the DMF being 1.1-1.2. In Bs-1 gamma R strain with intragenic suppressor of lexA gene the adaptive response efficiency was the same as in B/r (recA+lexA+) strain. There is no adaptive response to MMS in polA- strains. The adaptive response to MMS in E. coli is different from that to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methylnitrosourea (MNM), because in these two cases it is absolutely lexA-recA dependent. It is supposed that a partial recA-lexA dependence of the adaptive response to MMS in E. coli may be due to a specific MMS-induced lethal damage that induces an adaptive repair non-related to the system of recA-lexA-independent adaptive responses to MNNG and MNM. The presence of a plasmid of drug resistance pKM101 exerts no influence on the value, efficiency and recA-lexA-dependence of the adaptive response of E. coli to MMS.     

Effects of aflatoxin B1 and caffeine on viability in natural strains of Drosophila melanogaster

Thirty-five natural populations of Drosophila melanogaster from Virginia were tested for their degree of sensitivity to the carcinogen Aflatoxin B₁ (AFB₁) and to caffeine. Significant variations in sensitivity to each was demonstrated among these strains, but no correlation existed for relative degrees of sensitivity to AFB₁ or caffeine when strains were compared, although most strains were more severely affected by AFB₁. Eight of these strains were tested for the effects of simultaneous administration of AFB₁ and caffeine. Generally, caffeine demonstrated both a significant protective effect in preventing the high degree of egg-adult mortality produced by AFB₁ alone and a retardation of the significant decrease in body length shown by adults which survive in the AFB₁ treatments.     

The effect of caffeine on repair in Chlamydomonas reinhardtii: II. Interaction of repair systems

Survival following UV-irradiation of the two repair-deficient strains of Chlamydomonas reinhardtii, UVSE5 and UVSE6, was not affected by caffeine. Since caffeine causes increased survival in strains of this organism having normal recombination, these two mutant strains are considered to be recombination-deficient. The double-mutant strains UVSE1–UVSE4, UVSE1–UVSE5, UVSE1–UVSE6, UVSE4–UVSE5, UVSE4–UVSE6 and UVSE5–UVSE6 were isolated. These strains were exposed to UV-irradiation and in all but UVSE4–UVSE5, survival of the double-mutant strain was much lower than for any single-mutant strain. These results indicate that the altered gene products in UVSE1, UVSE5 and UVSE6 mutant strains are associated with different recombination-repair mechanisms.

All double-mutant strains were treated with caffeine following UV-irradiation. In all double-mutant strains containing a mutant USVE4 gene product, recombination repair was increased by caffeine.

On the basis of the data obtained, a scheme is proposed for the involvement of multiple repair systems in repair following UV-irradiation in C. reinhardtii.     

Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.     

DNA repair and sensitivity of mouse embryo fibroblasts to methyl methanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine

DNA repair synthesis and cytotoxicity were evaluated in early passage mouse embryo fibroblasts from five inbred strains (B10, CBA, C3H/A, DBA/2, BALB/c) and in BALB/3T3 IL-2 cells after the cultures had been treated for 3 h with methyl methanesulphonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In the presence of hydroxyurea, the incorporation of tritiated thymidine into the MMS- or MNNG-treated cells derived from B10, CBA, C3H/A or DBA/2 mice, was, at the concentrations used, significantly higher than into controls untreated with the mutagens. Under analogous experimental conditions there was no detectable DNA repair synthesis in two kinds of cells derived from BALB/c mice. MNNG was more cytotoxic to the cells derived from BALB/c mice than to those of the four remaining strains. The sensitivity of all kinds of early passage mouse fibroblasts to MMS was similar at each MMS concentration tested. Cloning efficiency of BALB/3T3 IL-2 cells exposed to MMS at the concentration of 10(-3) or 10(-4) M did not differ from that of untreated controls. The latter cells treated with MNNG at the concentration of 10(-4) or 2 X 10(-4) M did not develop colonies.     

Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid.

A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.