The mutational spectrum of procarbazine in Drosophila melanogaster.

The antineoplastic agent Procarbazine was tested for the induction of genetic damage in Drosophila melanogaster. The compound was administered to adult males by oral application. The following types of genetic damage were measured: (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. Procarbazine is highly mutagenic in causing recessive lethal mutations in all stages of spermatogenesis. In sperm a clear-cut concentration-effect relationship is not apparent, but in spermatids such a relationship is obtained for mutation induction at low levels of procarbazine exposure, while at high concentrations the induction of recessive lethals is not a function of concentration. A low induction of total sex-chromosome loss (X,Y) and dominant lethals was observed in metabolically active germ cells (spermatids), but procarbazine failed to produce well-defined breakage events, such as partial sex-chromosome loss (YL,YS) and II-III translocations. The results obtained in Drosophila melanogaster are discussed and compared with the mutational pattern reported in the mouse after procarbazine treatment.     

Genetic damage in domestic mice inhabiting in the areas with elevated background radiation

Genetic effects of irradiation in males of wild house mice which were caught in the region of Chernobyl Nuclear Power Station were studied. The dose rate on the ground surface varied from 0.04 to 200 mR/h of gamma-irradiation. The increasing yield of dominant lethal mutations was only observed in males from the most contaminated sector. Reciprocal translocations were observed in spermatocytes of mice at all the levels of contamination. The rate of reciprocal translocations was relatively low and increased linearly with the elevation of the dose rate. The extent of testis damages increased also, as the dose rate grew. The frequency of abnormal sperm heads, the yield of recessive lethal mutations, litter size and radiosensitivity of the first progeny were not changed, depending on the dose rate.     

Autosomal recessive lethal mutations in two mutator stocks of Drosophila ananassae.

The frequency of recessive lethals in the 2nd chromosome was examined in two mutator stocks of Drosophila ananassae, ca and ca; px. They are characterized respectively by possessing an extrachromosomal clastogenic mutator in males, and by the retrotransposon "tom", which induces Om mutability only in females. The frequencies of recessive lethal mutations in the 2nd chromosome among progenies from males and females of the ca; px stock are 0.35 and 0.34 percent, respectively. Similarity of these frequencies indicates that tom does not induce recessive lethals in females. In contrast to the ca; px stock, the frequency of recessive lethals in males of the ca mutator stock was estimated to be 1.54 percent for the 2nd chromosome. No visible mutants except Minutes were recovered. Some recessive lethals derived from ca stock males were associated with chromosomal rearrangements. Being consistent with its high rate of Minute mutation it was demonstrated that the ca clastogenic mutator also induced recessive lethals.     

Comparative study of the clastogenic efficiency of ethyl methanesulfonate and diethyl sulfate in Drosophila melanogaster mature sperm

Chromosome loss and translocation tests were carried out in Drosophila melanogaster sperm, stored in untreated females for up to 24 days, to compare the clastogenicity of ethyl methanesulfonate (EMS) and diethyl sulfate (DES). The sex-linked recessive lethal test was used as a "biological dosimeter" and the following results were obtained: The yield of 2-3 translocations induced by both mutagens increased steadily with storage, being significantly higher after EMS than after DES treatment. The frequencies of partial losses induced by EMS and DES were similar and increased with storage. With up to 11 days' storage, the frequency of complete loss induced by DES was higher than that induced by EMS and remained unchanged when storage was extended to 24 days. Complete loss induced by EMS increased significantly with further storage (12-24 days). With DES, complete (but not partial) loss was detected with a dose at which EMS failed to modify the control values. These data suggest that the lower recovery of II-III translocations after treatment with DES does not result from a low breaking capacity but from a diminished or delayed rejoining of the induced breaks. This could be due to a physiological impairment of the treated cells by the high toxicity of DES or to an actual lower rejoinability of the broken ends. The differential recovery of complete and partial losses after DES treatment further suggests that the mechanisms leading to the fixation of both types of damage are somehow different, and that processes intervening in the recovery of partial losses are less affected, or not at all, by the proposed reduced rejoining of chromosome breaks.     

Heritable translocation test on random-bred mice after prolonged triethylenemelamine treatment.

Heritable translocation and dominant lethal tests were conducted with random-bred Swiss albino male mice. The animals were provided drinking water containing triethylenemelamine (TEM) for 4 weeks, and were then mated for 3 successive weeks for analysis of dominant lethality and production of F1 progeny. Potential translocation carriers among F1 males were selected after two breedings and confirmed by cytogenetic analysis. Translocation heterozygotes were obtained in offspring of the TEM-treated groups, but not in the control groups. In F1 males produced from the first week of mating, the frequencies of translocations were 0, 1.78 6.2 and 10.0% for the control group and groups receiving TEM at 0.0125, 0.025 and 0.050 mg/kg/day, respectively, and in those produced from the third week of mating, the values were 0 and 2.1%, respectively, for the control group and the group receiving TEM at 0.050 mg/kg/day. F1 males from the second week of mating were not studied for the induction of heritable translocations. TEM-induced dominant lethality and heritable translocations were most prominent in the first week of mating after 4 weeks of treatment. In addition, heritable translocations appeared to be a more sensitive endpoint than dominant lethal mutations for the measurement of mutagenic effects of TEM.     

Comparison of lethal mutations of Drosophila melanogaster with D. simulans when detected by the attached-X method.

The purpose of this study was to evaluate the attached-X method compared with the standard Basc method, and, using this method, to find out whether the observed differences in genetic polymorphisms are related to differences in lethal mutation rates in D. melanogaster and D. simulans. When EMS-treated Drosophila melanogaster males are mated to untreated attached-X females, a decrease in the progeny sex ratio (male/female + male) is observed due to the induced lethal mutations on the X chromosome. The decrease in the frequency of male progeny were shown as the attached-X index. The expected male number is calculated from the control sex ratio. The difference between the expected and the observed male numbers, expressed as the ratio to the expected male number, defines the attached-X index. The index values for various EMS concentrations were compared to the lethal frequencies obtained by the standard Basc method for the same EMS treatments, and gave a highly positive correlation (gamma = 0.993, p < 0.01, d.f. = 2), thus providing an alternative method for evaluation of possible mutagens. The attached-X method was applied to D. simulans, of which natural populations are known to have relatively low genetic variation, and frequencies of the EMS-induced X chromosome lethal mutations were estimated and compared with those in D. melanogaster. The results indicate that D. melanogaster is slightly more sensitive in the sperm and spermatogonial stages, but less susceptible in the spermatid stage when compared with D. simulans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Instability in the ctMR2 strain of Drosophila melanogaster: role of P element functions and structure of revertants

Summary Simultaneous multiple transpositions and longterm genetic instability have been described in the ct ^MR2 strain of Drosophila melanogaster and its derivatives. This strain originated from a cross that was dysgenic in the P-M system. While spontaneous instability declined over 2 years, instability has been reactivated by backcross to the progenitor P element bearing strain MRh12/Cy. We show here using germline transformation that active P factor alone cannot mimic the effect of this cross, suggesting that MRh12/Cy contains some other activator. In addition, we have observed that ct ⁺ exceptional progeny arise in the F1 s well as the F2 generations. Molecular analysis of X chromosomes from some ct ⁺ progeny indicates that phenotypic reversion of the ct mutation can arise through two unrelated mechanisms.     

Mutagenicity studies in Drosophila melanogaster with Lannate 20

Lannate 20 a carbamate pesticide was evaluated for its mutagenicity in Drosophila melanogaster by the sex-linked recessive lethals and chromosome II-III translocation tests by continuous larval feeding. The 3 sublethal doses of 0.2, 0.4 and 0.6 microliter of Lannate per 100 ml of the food medium induced a significant (P less than 0.01) increase in the number of sex-linked recessive lethals over the controls. However, no translocations were observed either in the treated or the control series.     

Generation of Y-chromosome insertions into left arm of chromosome 2 of Drosophila melanogaster

An experimental procedure describing production of insertions of Y-chromosome material into autosomes of Drosophila is presented. Irradiated Y;2 translocations served as source material. The insertion selection scheme was based on the emergence of additional progeny classes in the case of independent segregation of the detached fragments of the Y chromosome and autosome. A total of seven insertions of Y-chromosome material into the left arm of chromosome 2, specifically in regions 29F, 34A, and 36B, were obtained. All insertions were lethal in the homozygous state and caused crossing-over suppression in the left arm of chromosome 2. In addition, these mutations induced the formation of loops between the chromocenter and the region of insertion, as well as breaks in one or both homologs, which are frequently observed in preparations of polytene chromosomes. The selection scheme suggested can be used to produce insertions in any region of Drosophila melanogaster chromosomes 2 and 3, for which the Y;2 translocations exist.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. II. Detection of qualitative differences between genetic damage induced by X-irradiation of mature spermatozoa in oxygenated and anoxic atmospheres through the use of the repair-deficient mutant mei-9a

Muller-5 males were irradiated with X-rays in nitrogen, in air or in oxygen (followed by nitrogen or oxygen post-treatments in the nitrogen and oxygen series) and were mated to females of a repair-proficient strain (mei+) or to those of a strain known to be deficient in excision repair of UV damage (in somatic cells). The latter strain, designated as mei-9a, is also known to be sensitive, in the larval stages, to the killing effects of UV, X-rays and to a number of chemical mutagens. The frequencies of sex-linked recessive lethals and autosomal translocations induced in the spermatozoa of males were determined and compared. The frequencies of sex-linked recessive lethals in the mei-9 control groups were consistently higher than in the mei+ groups. Irradiation in air or in nitrogen led to significantly higher yields of recessive lethals when the irradiated males were mated to mei-9 females, whereas, after irradiation in oxygen, the yields were similar with both kinds of female. No significant differences in the frequencies of reciprocal translocations were observed between the mei+ and mei-9 groups after irradiation of the males in nitrogen, in air or in oxygen. Likewise, no differential effects of the contrasting post-treatments (nitrogen versus oxygen), either for recessive lethals or for translocations, could be discerned. These results are considered to support the notion that the kinds of genetic damage induced in mature spermatozoa in air or in nitrogen are qualitatively similar (at least with respect to the component(s) that lead to the production of recessive lethal mutations), but clearly different when induced in an oxygen atmosphere. The enhanced yields of recessive lethals with mei-9 females (after irradiation of the males either in air or in nitrogen) has been interpreted on the assumption that the mei-9 mutant is also deficient for the repair of X-ray-induced, recessive lethal-generating premutational lesions. Possible reasons for the lack of differences between the mei+ and mei-9 groups with respect to translocation yields and for the absence of measurable differences in response between the contrasting post-treatments (after irradiation of the males in nitrogen) are discussed.     

Mutagenic effect of different types of irradiation on the sex cells of male mice. X. Frequency of recessive lethal mutations and reciprocal translocations in the spermatogonia of mice subjected to fractional gamma-irradiation]

The frequency of recessive lethal mutations and reciprocal translocations was investigated in spermatogonia of CBA male mice which were thrice gamma-irradiated at doses of 300 r with 28 days intervals. The rate of induced recessive lethals was estimated 1) by comparison of embryos survival between the irradiated and control groups in mating of the F1 males with their daughters, and 2) by estimation the frequency of males heterozygotes for recessive lethals in the first generation. In the first case the frequency of recessive lethals was 2,8 +/- 0,8-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete in the second case. The frequency of heterozygotes for reciprocal translocations in the first generations of males was 3,1 +/- 0,9-10(-5) per r per gamete.     

Increment kinetics of recessive lethal mutations and dominant lethals in offspring of Drosophila melanogaster on storage of methyl-methanesulfonate-treated sperm in females

The frequencies of sex-linked recessive lethal mutations in F1 males after feeding adult male Drosophila melanogaster with 0.25 and 0.5 mM methyl methanesulfonate (MMS) orally for 24 h increased approximately linearly with storage of the treated spermatozoa in females, whereas the number of hits of dominant lethals in the sperm after feeding 0.3 and 0.5 mM MMS increased approximately with the square of the storage time. Chromosome losses and mosaics in F1 males also increased with the dose of MMS to males, but their yields were too low to be analyzed quantitatively, only indicating a slight increase of chromosome loses and a slight decrease of mosaics with the time of storage of sperm. Maternal non-disjunctions (or chromosome losses), detected in F1 males, decreased with the dose of MMS to spermatozoa and their yield decreased with the time of storage of sperm of both MMS-treated and the control groups. A unitary model is proposed to explain the effect of storage on the dominant lethals and recessive lethal mutations.     

High Rates of Occurrence of Spontaneous Chromosome Aberrations in DROSOPHILA MELANOGASTER

Spontaneous mutations were accumulated for 40 generations in 140 unrelated second chromosomes with the standard gene arrangement. These were extracted from the same population by using the marked inversion technique, and the following findings were obtained: (1) In 42 out of the 140 chromosome lines, chromosome aberrations were detected by examining the salivary gland chromosomes: 40 paracentric and 15 pericentric inversions, 2 reciprocal translocations between the second and the third chromosomes, and 6 transpositions. (2) In 63 out of the 90 originally lethal-free lines, recessive lethal mutations occurred. (3) There were only 3 lines that acquired chromosome aberrations (inversions) with no lethal effects in the homozygous condition. (4) In a comparison of these results with those of the (CH), (PQ), and (RT) chromosomes in which no chromosome aberrations occurred after accumulating mutations for 22058 chromosome.generations (Yamaguchi and Mukai 1974), it was concluded that some of these 140 chromosomes carried a kind of mutator. (5) The frequency of mutator-carrying chromosome lines was estimated to be 0.66 on the basis of the distribution of the break-points on the chromosome lines and the frequency of lines that acquired neither recessive lethal mutations nor chromosome aberrations. Thus, the average number of breaks per mutator-carrying chromosome was estimated to be about 0.19/generation.On the basis of these estimates, the nature of the mutator factor was discussed.     

Effect of ethanol pretreatment on genetic damage induced by X-rays in Drosophila melanogaster sperm

Drosophila melanogaster males were treated with 96% ethanol for 45 min by means of soaked tissue paper placed at the bottom of regular culture vials before being exposed to 2 krad of X-rays. The use of ethanol was dictated by its high efficiency to scavenge hydroxyl radicals that play a substantial role in the indirect effect of ionizing radiation. The data obtained show that the frequency of sex-linked recessive lethals, reciprocal translocations and chromosome losses induced in postmeiotic cells were not reduced by ethanol pretreatment. Rather, in the combined treatments a significant increase in II-III translocations was observed in sperm. This effect declined in late and mid spermatids. Treatment with ethanol alone did not modify the frequencies of the genetic endpoints tested. It is tentatively suggested that: (i) ethanol or ethanol radicals impair the restitution of broken chromosome ends, thereby increasing the chances for rearrangement formation in the egg, or (ii) ethanol given prior to irradiation acts as a weak dose-modifying factor. If so, a slight increase in the effective dose could have resulted in a detectably higher frequency of translocations whose induction, unlike the other genetic damages investigated that increase linearly with dose, follows the slope of a 2-hit kinetic curve.     

Multigeneration exposure test of Drosophila melanogaster to ELF magnetic fields

Mutations, other than dominant lethals, were accumulated on wild type second chromosomes (+) of Drosophila melanogaster during exposure to 50 Hz sinusoidal alternating magnetic fields of 0.5 or 5 mT (rms) for 40 generations by the Curly/Plum(Cy/Pm) accumulation method. We maintained, for 40 generations under continuous exposure, each (+) chromosome as a heterozygote with (Cy) chromosome. Viability of the (+) chromosome was tested by sib-mating of (Cy/+) male and (Cy/+) female in a culture every 10th generation to obtain the homozygote. Viability indices, defined as twice the ratio of number of (+/+) flies to that of (Cy/+) flies plus 1 in the progeny of the test mating, also were calculated, which equaled 1.00 at the starting point. For the control and 0.5 and 5 mT exposed groups, percent frequencies of recessive lethal lines, defined as a line with (+/+) flies less than 0.3% in the test mating, were, respectively, 1.9, 0.9, and 2.9% (10th), 9.0, 4.9, and 9.5% (20th), 30.3, 22.9, and 30.4% (30th), and 39.9, 32.4, and 43.3% (40th generation). For the control and 0.5 and 5 mT groups, average viability indices, excluding lethals and markedly deleterious, were, respectively, 0.778, 0.796, and 0.752 (20th), 0.704, 0.698, and 0.694 (30th), and 0.669, 0.678, and 0.595 (40th generation). Their decreasing rates were 0.0054, 0.0059, and 0.0078 per generation. No significant difference was detected among the exposure levels in either the recessive lethal mutation frequency or the viability index. Bioelectromagnetics 19:335–340, 1998. © 1998 Wiley-Liss, Inc.     

Studies on mutagen-sensitive strains of Drosophila melanogaster VIII. Further data on differences between Canton-S and ebony strains with respect to maternal effects for the X-ray induction of autosomal translocations and ring-X chromosome losses in mature spermatozoa

The influence of the maternal genotype (Canton-S, proficient in the repair of X-ray-induced chromosome breaks and ebony, less proficient in this regard) on the recovery of X-ray-induced autosomal (II–III) translocations and ring-X chromosome losses in mature spermatozoa was studied. In the first series of experiments, males carrying appropriate markers on their second and third chromosomes were irradiated and mated to Canton-S or ebony females and the frequencies of II–III translocations were determined. In the second series of experiments, males carrying ring-X chromosomes were irradiated in N₂ or in O₂, mated to Canton-S or ebony females and the frequencies of XO males were determined; additionally, under similar gas-treatment and radiation conditions, the pattern of egg-mortality was also assessed.

The data on translocations show that the yields are higher with ebony than with Canton-S females; these and earlier results on dominant lethals and sex-linked recessive lethals support the interpretation that the maternal repair system in the ebony strain is less proficient and more error-prone than that of the Canton-S strain.

Those on the losses of ring-X chromosomes demonstrate that (i) the absolute yields of XO males are lower with ebony than with Canton-S females irrespective of whether the parental males are irradiated in N₂ or in O₂; (ii) the exposure-frequency relationships are all linear, but the slopes are higher when the males are irradiated in O₂ and are consistent with an oxygen-enhancement-ratio of about 1.5 and (iii) the relationships between the logarithm of egg-survival and XO male frequency are also linear, but the slopes for the O₂ groups are lower than those for the N₂ groups (slope ratios of 0.86–0.87).

The finding that at given survival levels, the XO frequencies are lower in the O₂ than in the N₂ groups of both the Canton-S and ebony series viewed in the context of the mechanisms that have been postulated to explain the loss of ring-X chromosomes in irradiated mature spermatozoa permits the following interpretation for the observed results: (i) a higher proportion of potential XO zygotes is lost through dominant lethality in the O₂ groups than in the N₂ ones presumably because the chromosome breaks induced in O₂ are qualitatively different in the sense that they have higher probability to undergo reunions relative to restitution, compared with breaks induced under anoxia and (ii) this leads to lower than expected oxygen-enhancement ratios (i.e., expected on the basis of published data on sex-linked recessive lethals, another kind of genetic damage which shows a linear exposure-frequency relationship).     

Delayed Formation of Chromosome Aberrations in Mouse Pachytene Spermatocytes Treated with Triethylenemelamine (Tem)

Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis–metaphase I stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis–metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed—a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis.     

Genetic Damage at Specific Gene Loci in Drosophila with Betatron X-Rays

The mutation rate was determined for mature sperm at eight specific gene loci on the third chromosome of Drosophila melanogaster using the low ion density radiations of 22 Mev betatron X-rays. A dose of 3000 rads of betatron X-rays produced a mutation rate of 4.36 x 10^-8 per rad/locus. Among the mutations observed, 66% were recessive lethals and 34% viable when homozygous. Only one of the 24 viable mutations was associated with a chromosome aberration. Among the 47 recessive lethals, no two-break aberrations were detected in 48.9% of the lethals, deletions were associated with 42.2%, inversions with 6.7% and translocations with 2.2%.—When these genetic results are compared to those for 250 KV X-rays, the mutation rate for betatron treatments was slightly lower (.76), the recessive lethal rate among induced mutations was higher, and the chromosome aberrations among lethal mutations were slightly lower than with 250 KV X-rays. Although the two types of irradiations differ by an ion density of approximately ten, the amount and types of inheritable genetic damage induced by the two radiations in mature sperm were not significantly different.     

Evidence of an essential difference between point mutations and chromosome breaks induced by triethylene melamine inDrosophila spermatozoa

Summary Storing of triethylene melamine-treated mature spermatozoa in untreated females was found to result in increased frequencies of both sex-linked recessive lethals and translocations involving the Y, II and III chromosomes. Frequencies of these mutations in effectively unstored spermatozoa were determined from progenies produced using sperm 2–4 days after treatment. The increase in translocation frequencies was on the order of 12-fold in progenies from sperm utilized 11–13 days after treatment when the sperm were stored at 25°C, and 3- to 6-fold when comparable sperm were stored at 12.5°C. Consistent but much smaller increases in frequencies of sex-linked lethals were found, with the increase in lethals tending to be correlated with relative increase in translocation frequency in a given experiment. On the assumption that sex-linked lethals related to chromosome breakage would be expected to increase in frequency in the same proportion as do translocations, approximate agreement was obtained when the proportions of breakage-related lethals among unstored lethals were estimated from the data in the four experimental series. The data are thus consistent with the hypothesis that chromosome breaks but not point mutations are realized during storage of treated spermatozoa. Possible interpretations of a differential effect of storage on treated chromosomes are discussed.Studies carried out while the author was a guest investigator at the Institute of Animal Genetics on sabbatical leave from the University of Minnesota.     

Chromosomal principle of radiation-induced F1 sterility in Ephestia kuehniella (Lepidoptera: Pyralidae).

A dose-response analysis of chromosomal aberrations was performed in male progeny of gamma-irradiated males in the flour moth, Ephestia kuehniella. For comparison, several female progeny from each dose level were examined. Aberrations were detected on microspread preparations of pachytene nuclei in the electron microscope and classified according to pairing configurations of synaptonemal complexes (SCs). Fragmentation and various translocations were the most numerous aberrations, whereas interstitial deletion and inversion were rare. At 100 Gy, relatively simple multiple translocations were found. Multiple translocations showing complicated configurations occurred at 150 and 200 Gy, and their number increased with the dose. In males, the mean number of chromosomal breaks resulting in aberrations linearly increased with the dose from 8.4 to 16.2 per nucleus. In females, this value achieved a maximum of 11.2 breaks/nucleus at 200 Gy. Three factors were suggested to contribute to the reported higher level of F1 sterility in males than females: (i) survival of males with high numbers of breaks, (ii) crossing-over in spermatogenesis but not in the achiasmatic oogenesis, and (iii) a higher impact of induced changes on the fertility of males than females. It was concluded that translocations are most responsible for the production of unbalanced gametes resulting in sterility of F1 moths. However, F1 sterility predicted according to the observed frequency of aberrations was much higher than the actual sterility reported earlier. This suggests a regulation factor which corrects the predicted unbalanced state towards balanced segregation of translocated chromosomes.