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.     

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.     

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.     

Involvement of separate pathways in the repair of mutational and lethal lesions induced by a monofunctional sulfur mustard.

The mutagenic and lethal effects of a monofunctional sulfur mustard, 2-chloro-ethylethylsulfide (CEES), have been studied in a number of repair deficient variants of Escherichia coli K12, B/r and B. The results indicate that CEES induces a (pre)mutational lesion which is subject to Uvr+-excision-repair. Extensive CEES-induced mutagenesis can occur in exrA- uvrA- and recA- uvrB- variants suggesting that the majority of the mutations in Uvr-bacteria do not arise from error-prone repair. These findings are similar to results previously reported with a volatile degradation product of captan and with ethyl methanesulfonate (EMS) but differ from those reported with methyl methanesulfonate (MMS). It is hypothesized that CEES alkylates guanine at the O-6 position (R-O-6-G) and that this R-O-6-G which is Uvr+-excisable is directly mutagenic by producing G-C to A-T transitions during replication. Reduced levels of induced mutation frequencies observed in an endonuclease II-deficient variant lead us to postulate that, in constrast to Uvr- bacteria, CEES-induced mutation in wild-type cells arise from error-prone repair of apurinic sites. Analysis of the lethal actions of CEES indicates that the lesion produced is largely unexcisable by the Uvr+ system. Host-cell reactivation of CEES-treated TI bacteriophage shows that the production of the (pre)ethal lesion is dependent on both the initial dose and post-treatment incubation. The efficient repair of the (pre)ethal lesion requires both endonuclease II and polymerase I. Moreover, deficiencies of these two enzymes rendered bacteria more sensitive to the cytotoxic action of CEES. It is postulated that the lethal mechanism of CEES involves: (I) alkylation at the N-3 position of adenine and the N-7 position of guanine; (2) spontaneous depurination of these alkylated bases; and (3) production of apurinic sites which are lethal unless repaired by the endonuclease II-polymerase I excision-repair system.     

Ethylation pf DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals

The molecular dosimetry of ethyl methanesulfonate (EMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 200 mg/kg of [³H]EMS and the ethylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 2-week period. The ethylations per sperm head closely paralleled the dominant-lethal frequency curve for EMS, reaching a maximum of 5 to 6.5 million ethylations per vas sperm head at 8 to 10 days after treatment. Ethylation of sperm DNA was greatest at 4 h after treatment, with 5.7 ethylations/10⁵ deoxynucleotides, and gradually decreased to 2.2 ethylations/10⁵ deoxynucleotides at 15 days after treatment. The ethylation of sperm DNA did not increase in the germ-cell stages most sensitive to EMS, ans was not correlated with the dominant-lethal frequency curve for EMS. However, ethylation of sperm protamine did increase in the germ-cell stages most sensitive to EMS, and showed an excellent correlation with the incidence of dominant lethals produced by EMS in the germ cells.A model is presented to explain, at a molecular level, how dominant lethals may be induced in mouse germ cells by EMS. Ethylation by cysteine sulfhydryl groups contained in mouse-sperm protamine could block normal disulfidebond formation, preventing proper chromatin condensation in the sperm nucleus. Stresses in the chromatin structure could then eventually lead to chromosome breakage, with resultant dominant lethality.     

Chromosome damage in the bone marrow of mice treated with the methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea in the presence or absence of caffeine, and its relationship with thymoma induction.

A single dose (0.8 mmole/kg) of N-methyl-N-nitrosourea (MNUA) causes significantly more chromosome damage in the bone marrow of mice than a dose of equal toxicity to the animals, (1.1 mmole/kg) of methyl methanesulphonate (MMS) 6, 24 and 48 h after treatment. At these doses both agents alkylate bone-marrow DNA to similar extents, but only MNUA induces thymic lymphomata. The greater chromosome-damaging effects of MNUA are ascribed to the known differences in the pattern of DNA alkylation by each agent, in particular the much higher levels of O-6 methylguanine and phosphotriesters produced by MNUA. The greater chromosome-damaging effect of MNUA may account for its higher toxicity to the bone marrow which in turn may be a significant factor in the induction of thymomata. The enhancement by caffeine of chromosome damage seen particularly 48 h after MMS-treatment suggests that post-replication repair protects cells from the effects of DNA-methylation in vivo.     

Effect of mutagens, chemotherapeutic agents and defects in DNA repair genes on recombination in F' partial diploid Escherichia coli.

The ability of mutagenic agents, nonmutagenic substances and defects in DNA repair to alter the genotype of F' partial diploid (F30) Escherichia coli was determined. The frequency of auxotrophic mutants and histidine requiring (His-) haploid colonies was increased by mutagen treatment but Hfr colonies were not detected in F30 E. coli even with specific selection techniques. Genotype changes due to nonreciprocal recombination were determined by measuring the frequency of His- homogenotes, eg. F' hisC780, hisI+/hisC780, hisI+, arising from a His+ heterogenote, F' hisC780 hisI+/hisC+, his1903. At least 75% of the recombinants were homozygous for histidine alleles which were present on the F' plasmid (exogenote) of the parental hetergenote rather than for histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which block DNA synthesis and a defective DNA polymerase I gene, polA1, were found to increase the frequency of nonreciprocal recombination. A defect in the ability to excise thymine dimers, uvrC34, did not increase spontaneous nonreciprocal recombination. However, UV irradiation but not methyl methanesulfonate (MMS) induced greater recombination in this excision-repair defective mutant than in DNA-repair-proficient strains. Mutagenic agents, with the exception of ethyl methanesulfonate (EMS), induced greater increases in recombination than the chemotherapeutic agents or the polA1 mutation. EMS, which causes relatively little degradation of DNA, was more mutagenic but less recombinogenic than MMS, a homologous compound ths that inhibition of DNA occurring single-stranded regions in replicative intermediates of the DNA. Mutagens which cause the rapid breakdown of DNA may, in addition, introduce lesions into the genome that increase the number of single-stranded regions thus inducing even higher frequencies of recombination.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.