Chromosome aberrations induced by gamma radiation in the somatic cells of a radiosensitive mutant line of Drosophila melanogaster

Chromosome aberrations induced by gamma-rays in ganglia cells of Drosophila melanogaster larvae have been studied. Two strains of Drosophila were used: radiosensitive mutant rad (2) 201G1 and normal strain. It has been shown that the frequency of cells with chromosome aberrations in radiosensitive larvae is much more than in normal larvae after gamma-irradiation. The ratio of chromosome and chromatid deletions number to the number of exchange type aberrations is the same for both strains. The kinetics of chromosome aberrations induced in rad-larvae is similar to the normal one. The conclusion has been made that the realization of rad (2) 201G1 mutation takes place on the cell level.     

Radiosensitive Drosophila lines. VIII. The effect of the mus (201)Gl gene of Drosophila melanogaster on the sensitivity of the flies to ultraviolet rays in the imago stage

The effect of UV-light on survival of adult flies of the mutant line mus(2) 201G1 sensitive to methyl methanesulfonate (MMS) was studied. One day old flies were irradiated by UV-light and the survival was determined depending on the UV dose, the genotype and the sex of irradiated flies. High sensitivity of mutant flies was shown in the imaginal stage when division of somatic cells was absent. Moreover, the differences in sensitivity to UV-light were observed depending on the sex of flies. We suggest that the mutation mus(2)201G1 blocking the excision repair affects undivisional cells of the imaginal stage of Drosophila melanogaster.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Radiosensitive strains of Drosophila. XII. Realization of the effect of mutation rad(2)201G1 at the cell and organism levels

Two effects of gamma-rays were studied on radiosensitive mutant rad(2)201G1 and wild type strain rad+ of Drosophila: the rate of radiation-induced chromosome aberrations in somatic cells and lethality of individuals irradiated at different stages of preimaginal development. It has been shown that mutant strain is characterized by the increased rate of chromosome aberrations in somatic cells and lethality of developing flies. Control strain rad+ is characterized by more complicated relationship between the effects analyzed. The results obtained are discussed in connection with the action of rad(2)201G1 gene on repair of genetic damages and with existence of postradiation compensation mechanisms intrinsic in development of multicellular organisms.     

Sensitivity of larvae of Drosophila melanogaster mutant mus(2)201G1 to various analgesics

Sensitivity of larvae from mutagen-sensitive mus(2)201G1 mutant of Drosophila melanogaster to analgin, amidopyrine and antipyrine (4-64 mkmol/ml of medium) was studied. Relative frequency of flies homozygous for this mutation after combined development of homozygous and heterozygous flies in the medium with the analgetic was used as a criterion for larval sensitivity. It is shown that sensitivity of homozygotes was significantly higher than that of heterozygotes to all three analgetics. Relative sensitivity of homozygotes increased with the elevation of drugs concentration. The data obtained demonstrate the mutagenicity of the analgetics tested for Drosophila.     

Analysis of the Chromosome Aberrations Induced by X-Rays in Somatic Cells of DROSOPHILA MELANOGASTER

A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.-The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chromatid aberrations. They also display a greater frequency of spontaneous aberrations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G(2) and G(1). These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferentially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translocations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.     

Somatic cell mutagenicity in Drosophila melanogaster in comparison with genetic damage in early germ-cell stages

With the intention of assessing the general performance, sensitivity and the underlying mechanisms of somatic cell mutagenicity assays in Drosophila, a study was undertaken to compare the effectiveness of 5 procarcinogens and 4 direct-acting agents in the white/white-coral eye mosaic assay (SMART) with their activity in early (premeiotic) male and female germ-cell stages, after exposure of Drosophila larvae. The outcome indicated a lack of agreement in the results from recessive lethal assays (SLRL) in comparison with the somatic mutation and recombination test (SMART). The procarcinogens 2-naphthylamine (NA), 3-methylcholanthrene (MC), 9,10-dimethylanthracene (DA) and 7,12-dimethylbenz[a]anthracene (DMBA), and the direct-acting mutagens bleomycin (BM), methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), were quite efficient in producing somatic recombination and mutations in white/white-coral larvae, as opposed to only weak effects in early germ-cell stages. 2-Acetylaminofluorene (2AAF) showed marginal effects in both germ cells and somatic tissue after exposure of female larvae, but was inactive in testis. The discrepancy in mutational response between somatic cells and premeiotic germ cells is most impressive for MMS and BM. There is sufficient evidence for attributing a good sized proportion of the encountered variation to efficient error-free DNA repair of premutational damage and to segregational elimination during meiosis of deleterious mutations: (1) The efficient point mutagen ENU was the but one agent producing high levels of viable genetic alterations in early germ cells and in somatic cells. A similar behaviour was previously described for diethylnitrosamine, which ethylates DNA in the same fashion as ENU. (2) In early germ-cell stages of mei-9L1 male larvae, MMS induced multiple mutations (putative clusters) at a low dose differing by a factor 20-40 from those needed to produce an equivalent response in repair-competent strains. This is consistent with the concept of an active excision repair in premeiotic cells. (3) In the case of EMS, next to DNA repair, germinal selection seems to restrict the realization of EMS-induced genetic damage in premeiotic cells. (4) Bleomycin-induced chromosome aberrations caused high mortality rates in males (hemizygous for an X-chromosome) but not in females. MMS and BM, agents known to show preference for chromosome aberration induction, produced 3-6-fold higher rates of somatic mutational events (SME) in female genotypes as compared with the other sex.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA reparation parameters during epigenetic effect of the rad201(G1) mutation in Drosophila

Cell estimates of genetic damage repair were obtained to characterize the epigenetic effect of the rad201(G1) mutation. The estimates included morphological defects (malformations); the frequency of chromosome aberrations in somatic cells; and somatic mosaicism, reflecting double-strand break repair via conversion. The range and frequency of malformations significantly differed between the rad201(G1) epigenetic effect and irradiation. A high pupal lethality, detected upon P-element mobilization, was not associated with an increase in the frequency of cells with chromosome aberrations, while somatic mosaicism was far greater. The results are discussed in the context of differences between radiation and P-element mutagenesis.     

Influence of mus201 and mus308 mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster.     

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)     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

Cell estimates of genetic damage repair under the epigenetic effect of the rad201(G1) mutation in Drosophila

Cell estimates of genetic damage repair were obtained to characterize the epigenetic effect of the rad201(G1) mutation. The estimates included morphological defects (malformations); the frequency of chromosome aberrations in somatic cells; and somatic mosaicism, reflecting double-strand break repair via conversion. The range and frequency of malformations significantly differed between the rad201(G1) epigenetic effect and irradiation. A high pupal lethality, detected upon P-element mobilization, was not associated with an increase in the frequency of cells with chromosome aberrations, while somatic mosaicism was far greater. The results are discussed in the context of differences between radiation and P-element mutagenesis.     

Effect of caffeine on the frequency of radiation-induced chromosome aberrations at different stages of the cell cycle

The effect of caffeine on the frequency of chromosome aberrations in human lymphocytes irradiated at different stages of the cell cycle was studied. Caffeine appeared to nearly double the frequency of chromosome aberrations induced by irradiation at S and G2 stages and did not influence the effect of irradiation at G0 and G1 stages.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Radiosensitive Drosophila strains. IX. An analysis of the fertility and frequency of dominant lethal mutations in gamma-irradiated females of the mutant rad(2)201G1 strain

Fertility and frequency of gamma-induced dominant lethals in female oocytes have been studied in a strain of Drosophila melanogaster carrying rad(2)201G1 mutation and in the wild type strain. It was shown that oocytes of the mutant strain exhibited the higher sensitivity during the whole period of oogenesis, as compared to those of the wild type flies. The strongest influence of rad(2)201G1 mutation on the frequency of dominant lethals and fertility was observed.     

Effects of hyperthermia on radiation-induced chromosome breakage and loss in excision repair deficient Drosophila melanogaster

Hyperthermia increased radiosensitivity with respect to gamma-ray induced chromosome loss and breakage in all stages of spermatogenesis in the wild type Oregon R strain of Drosophila melanogaster, whereas hyperthermia increased radiosensitivity to a lesser extent in cn mus (2) 201D1, an excision repair mutant with 0 per cent excision capacity and in mus (3) 308D1, a strain with 24 per cent excision capacity. The differences in hyperthermia-induced radiation sensitivity between the excision repair mutants and the wild strain may be due to the hyperthermia affecting the excision repair mechanism, suggesting that one of the possible mechanisms involved in hyperthermia-increased radiosensitivity is an effect on excision repair.     

Effect of methylmethane sulfonate-sensitive mutations on nondisjunction and loss of sex chromosomes in Drosophila males

The MMS-sensitive mutants mus(1) 120M1 and mus(1) 121M1 of Drosophila melanogaster were investigated regarding their effects on spontaneous and X-ray induced (2000 R) aneuploidy in male germ cells, during different stages of spermatogenesis. In matings of males carrying mus mutation and a doubly marked Y-chromosome (BsYy+) with repair proficient y f females, the frequencies of partial loss, nondisjunctions and especially complete loss were significantly higher than in the control. Apparently, MMS-sensitive mutants deal with meiotic processes and maintenance of chromosome structural stability both in females and in males, in somatic and germ cells.     

Mutagen-sensitive mutants in Drosophila melanogaster: effects on premutational damage.

Drosophila melanogaster males from a Basc stock were mutagenized with either X-rays, ethyl methanesulfonate (EMS), or nitrogen mustard (HN2). Groups of identically treated males were crossed to different types of female. Sex-linked recessive lethals were scored as a genetic end point. The females used were homozygous for X-chromosomal mutations (mus(1)101D1, mus(1)104D1, mei-9 or mei-41D5) which lead to defective DNA repair and which increase the mutagen sensitivity of larvae. Females from a white stock with normal DNA repair capacities served as controls. The premutational lesions induced in mature sperm are only processed after insemination by the maternal enzyme systems present in the oocytes. Differences in the efficiency of the processing of lesions can lead to maternal effects on the frequency of mutations recovered from mutagenized sperm. It was found that, with the exception of mus(1)104D1, all mutants analysed significantly modify the mutation fixation of one or more types of premutational lesions. The most drastic effect is found with the mus(1)101D1 stock in which HN2-induced DNA cross-links do not lead to sex-linked recessive lethals. It is assumed that mus(1)101D1 is defective in an early step of DNA cross-link repair. Our first set of data clearly demonstrates that the study of maternal effects in Drosophila is an efficient tool to analyse the in vivo function of repair mutations on chemically induced mutagenesis.     

Differential cell mutability during oogenesis and exposure to ethyleneimine and ethylmethane sulfonate in different Drosophila melanogaster lines

The rate of recessive sex-linked lethal mutations (RLM) was estimated by brood pattern method at different stages of oogenesis, initially, in the wild-type R-86 strain of Drosophila melanogaster after treatment with EI and EMS. The former which is known to induce dominant lethals in mature oocytes of the 14th stage with a high frequency was equally effective in inducing RLM in oocytes of different age and in oogonia. EMS which does not induce dominant lethals when used as vapour was shown to increase RLM frequency in mature fraction of oocytes (the 14A stage only). Similar type of different mutability was found in mutagen-sensitive strain mus-201G1 and in the control 3-4 strain having the same genetical background as mus mutation. Female germ cells of mus-201G1 strain appeared to have a higher mutability in the case of EI, though no differences in mutability between these strains after EMS treatment were registered. The data are discussed in view of the specificity of primer damages occurring as a result of comparable mutagens action and participation of different repair systems in elimination of these damages.     

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.