Neutrons and X-rays, comparative studies with Drosophila melanogaster. 2. Sex-chromosome loss and partial loss, evidence for the induction of chromatid aberrations in spermatozoa

Losses and duplications of BSY y+-chromosome markers were induced by irradiation of spermatozoa with either 0.5-MeV neutrons or 100-kV X-rays. These 2 types of radiation are known to induce significantly different ratios of double:single strand breaks in DNA. Exceptional progeny were grouped into 3 categories; no Y marker, one Y marker, and Y marker duplications + mosaics. The last combination consisted of exceptions derived from only chromatid-type rearrangements. All other classes of exceptions may be derived from either chromatid- or chromosome-type rearrangements. Doses of 15 Gy neutrons and 27 Gy X-rays induced identical frequencies of exceptional progeny, giving an RBE of 1.8. The ratios of the 3 classes of exceptions were similar for both types of radiation. This observation can be interpreted as indicating that, under the conditions used here, chromosome and chromatid rearrangements are not derived directly from double and single DNA strand breaks, respectively.     

Studies on storage effects and the negative synergism between trenimon and X-rays in Drosophila

The experiments reported in this paper were designed to answer some questions relating to the augmenting effect of storage of sperm in the inseminated female, on the frequency of translocations in spermatozoa treated with the trifunctional alkylating agent trenimon. To see whether, upon storage, more chromosome breaks become available for interaction, sperm cells that had been treated with trenimon in the male were exposed to X-irradiation before or after 6 days storage in the female. The data of the first experiment indicated that in unstored sperm the translocation yield after treatment with both trenimon and X-rays, was lower than that expected on the basis of additivity of yields of the single treatments. The negative synergism between trenimon and X-irradiation has been confirmed in further experiments with both translocations and dominant lethals. The latter finding does not support an interpretation in terms of selective elimination of translocationsby cell death. Following storage, the translocation frequencies increase and after combination of trenimon and X-rays, yields corresponding to additivity of frequencies with single treatments are observed.To study whether changes in the maternal repair system contribute to the storage effect, trenimon-treated males were mated with aged females and the frequencies of translocations were determined. It was found that these frequencies were similar to those in young (unstored) females; this result suggests that the possibility raised above is unlikely.     

Mutagenic activity of ethylenimine in Neurospora crassa

The mutagenic activity of the monofunctional alkylating agent ethylenimine (EI) was tested with the adenine-3 (ad-3) system in a two-component heterokaryon of Neurospora crassa. The results of forward-mutation experiments showed that EI is a potent mutagen in N. crassa.Genetic analysis of EI-induced ad-3 mutants showed that the frequencies of leakiness, allelic complementation, and non-polarized complementation patterns are similar to those of ad-3 mutants induced by other alkylating agents. It seems, therefore, that in addition to multilocus deletions (which occur at low a frequency), EI-induced mutations probably include base-pair substitutions, frameshift mutations, and other types of intragenic alterations.     

Evidence of an essential difference between the genetical effects of mono- and bi-functional alkylating agents

Summary Following treatment ofDrosophila sperm with ethylene imine (EI) and triethylene melamine (TEM), the treated sperm were stored for six days in the inseminated females. It was found that after storage, the TEM translocation/lethal ratio rose steeply, due to a large increase in translocation frequency accompanied by a much smaller increase in sex-linked lethal frequency. The ratio for EI, however, remained constant before and after storage. This shows an essential difference between mono- and poly-functional alkylating agents, which is presumably related to the mechanism by which they produce chromosome breaks. The hypothesis is put forward that the storage effect might be the main reason for the much greater effectiveness of polyfunctional agents in carcinostasis.     

X-ray induction of dominant lethals in late spermatids and mature spermatozoa of Drosophila melanogaster: the role of oxygenation

The role of oxygenation in determining the sensitivity to the induction of dominant lethals was examined in late spermatids and mature spermatozoa of Drosophila melanogaster. 0–2-h-old or 7-day-old Oregon-K males were irradiated with different X-ray exposures in nitrogen, air or in oxygen and the frequencies of dominant lethals induced in these stages were studied. The results obtained confirm and extend Sobels' earlier observations and the interpretation derived therefrom namely, that under normal conditions in air, mature spermatozoa are characterised by a higher degree of oxygenation than late spermatids and this difference is sufficient to explain the differential response of these stages. Similar Oxygen-Enhancement-Ratios(OERs) (of about 2) were obtained for both the cell stages. The present data also revealed that late spermatids are significantly less sensitive than mature spermatozoa to the X-ray-induction of dominant lethals in a nitrogen atmosphere. A plausible mechanism is suggested to explain this observation.     

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.     

Liquid storage of turkey semen: changes in quality parameters, lipid composition and susceptibility to induced in vitro peroxidation in control, n-3 fatty acids and alpha-tocopherol rich spermatozoa

The study considered two major aims: (a) to measure the changes in quality parameters, lipid composition and antioxidant activity occurring in turkey spermatozoa during liquid storage; (b) to determine if the enrichment of sperm in n-3 fatty acids and alpha-tocopherol affect sperm survival during storage. Turkey breeders were fed a control diet or an Omega3 diet enriched with fish oil and alpha-tocopheryl-acetate. Ejaculates were pooled (5ejaculates/pool; 4pools/treatment) and stored in vitro for 48h at 4 degrees C. Viability, motility, susceptibility to induced peroxidation and alpha-tocopherol content were measured in spermatozoa; lipid and phospholipid fatty acid composition were measured in spermatozoa and seminal plasma. The proportion of motile and viable spermatozoa significantly decreased, and the proportion of dead spermatozoa significantly increased. The susceptibility of turkey spermatozoa to induced peroxidation also significantly increased during storage. The enrichment of turkey spermatozoa with n-3 long chain PUFA and vitamin E by dietary treatment did not prevent the negative effect of storage on sperm quality and sensitivity to induced in vitro peroxidation; however, it was efficient in partially prevent the increase of sperm death, therefore the proportion of dead spermatozoa was higher in control (37.4%) compared to treated spermatozoa (31.7%) after 48h liquid storage. Major changes were recorded in the lipid composition of turkey spermatozoa during liquid storage in both experimental dietary groups, whereas no significant changes were measured in seminal plasma. In spermatozoa, a great loss in the phospholipid and free cholesterol content was measured. Moreover, the loss in total sperm phospholipid was associated to a peculiar and selective decrease in the bounded fatty acids: saturates and monounsaturates were greatly reduced and polyunsaturates did not change. As a consequence, the polyunsaturated to saturated fatty acid ratio increased during 48h liquid storage. The observed changes in the lipid and phospholipid-bound fatty acid composition of turkey spermatozoa occurring during liquid storage might be related to different events and have been discussed.     

The relation between chemically induced sister-chromatid exchanges and chromatid breakage.

Studies of classical chromosome aberrations and sister-chromatid exchanges (SCES) suggest independent mechanisms for the two events despite some common features. Examination of chromosome breakage caused by X-rays, visible light, and viruses has shown that few chromatid breaks are accompanied by SCEs at the sites of breaks. No similar observations were available for chemically induced breaks, but it has been reported that rat chromosomes exposed to dimethylbenzanthracene (DMBA) contained a preponderance of both aberrations and SCEs in certain specific regions, implicating a common process in their formation. These conclusions were drawn from a comparison of breaks induced in vivo with SCEs induced in vitro. However, we used 7 chemical mutagens to induce both chromatid breaks and SCEs in "harlequin" chromosomes of cultured rat and Chinese hamster ovary (CHO) cells and found that 25% of the 914 breaks scored were associated with SCEs. The proportion of breaks accompanied by SCEs is related to the overall SCE frequency and falls into the range predicted on the basis that breaks and SCEs occur independently. The reported association between sites for SCEs and aberrations also reflects secondary factors, such as induction of SCEs and aberrations during DNA synthesis in late replicating regions of the chromosomes.     

Factors affecting the quantitation of dose-response curves for mutation induction in V79 Chinese hamster cells after exposure to chemical and physical mutagens.

Using four common mutagens, ethyl methanesulphonate (EMS), methyl methanesulphonate (mms), uv, and X-irradiation, the relationship between dose of mutagen, cellular lethality and frequency of 8-azaguanine resistant colonies in V79 Chinese hamster cells has been examined. Several factors affecting the recovery of mutants including inter and intra-clone metabolic co-operation have been quantitated and their influence on survival response curves examined. Induced mutant frequencies were assayed by two methods in situ, and after replating. After exposure to X-rays, MMS and UV a significantly higher frequency of mutants was observed in replated experiments as compared with the in situ situation, at all survival levels assayed. With EMS, an increment on replating was observed only at high survival levels. The replating data suggest that two types of azgr colonies are produced, i.e. those which contain only azgr cells and those which, due to damage segregation, contain a mixture of azgr and azg8 cells. These mixed colonies appear to be lost by metabolic co-operation when mutation frequencies are assayed in silu. The proportion of mixed to homogeneous colonies differs with different mutagens. Taking into account such factors, EMS and UV irradiation were similarly mutagenic at a given survival level, but at equitoxic doses, fewer mutants were recovered after exposure of V79 cells to MMS and X-rays.     

A comparison of mutation induction by 14 MeV fast neutrons and 137 Cs -rays in meiotic spermatocytes in the silkworm

Barley seeds were treated with methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), stored at 15% water content and washed for 16–24 h. These treatments resulted in an increase of toxic and genetic effects. In teh DNA of embryos of such stored MMS- and EMS-treated seeds, a strong enhancement of the amount of single-strand breaks and/or alkali-labile sites took place. In contrast, the amount of alkylated sites, particularly of 7-methylguanine, was somewhat lower. It can be that the depurination and/or backbone breakage, which proceeds during the storage period, is responsible for the enhancement of toxic and genetic effects, whereas the influence of the alkylation of DNA during the storage period by the unreacted residual mutagen is negligible.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

Mechanisms for chromosomal aberrations in mammalian cells

Experimental evidence is presented for the involvement of DNA double-strand breaks in the formation of radiation-induced chromosomal aberrations. When X-irradiated cells were post-treated with single-strand specific Neurospora crassa endonuclease (NE), the frequencies of all classes of aberration increased by about a factor 2. Under these conditions, the frequencies of DNA double-strand breaks induced by X-rays (as determined by neutral sucrose-gradient centrifugation), also increased by a factor of 2. The frequency of chromosomal aberrations induced by fast neutrons (which predominantly induce DNA double-strand breaks) was not influenced by post-treatment with NE. Inhibition of poly(ADP-ribose) polymerase, an enzyme that uses DNA with double-strand breaks as an optimal template, by 3-aminobenzamide also increased the frequencies of X-ray-induced chromosomal aberrations, which supports the idea that DNA double-strand breaks are important lesions for the production of chromosomal aberrations induced by ionizing radiation.     

The induction of chromosome aberrations in mouse dictyate oocytes by X-rays and chemical mutagens.

Oocytes were collected from female mice and matured in vitro to Metaphase I during the first or third week after treatment with a dose of 400 rad X-rays, 1.6 mg/kg triethylenemelamine (TEM) or 75 mg/kg isopropylmethanesulphonate (IPMS). In week 1 the mean number of oocytes per female was similar for all treatments but in week 3 irradiated females yielded fewer oocytes than the chemically treated or control females. In week 1 the proportion of oocytes maturing was smaller in irradiated females than in the other groups but in week 3 was similar in all groups.Structural chromosome aberrations, scored in the Metaphase I oocytes, were of the chromatid or isochromatid type and involved gaps, breaks, fragments, intrachanges and interchanges. Aberration frequency did not increase with time after either of the chemical mutagens but after irradiation was higher in the third week than in the first week. The aberration yield from IPMS-treated females was similar at both sampling times, while a lower yield was recorded in week 3 following TEM treatment than in week 1.     

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.     

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).     

Heritable translocation test and dominant-lethal assay in mice with methyl methanesulfonate.

A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F1 males varied between 2 and 99%. Transmission of partial sterility and translocations was confirmed in the F2 generation. There were no partially sterile or sterile males among the 245 controls.     

Caffeine-induced enhancement of chromosome damage in human lymphocytes treated with methyl-methanesulphonate, mitomycin C and X-rays

Two hypothese have been put forward in the literature to explain the synergistic effect of caffeine with several mutagens: (1) binding of caffeine to DNA, and (2) inhibition of DNA repair.Autoradiographic studies with ³H- and ¹⁴C-labelled caffeine did not support the binding hypothesis. Caffeine enchanced in a synergistic way the amount of chromatid breaks and exchanged induced in human lymphocytes with methyl-methanesulphonate (MMS), mitomycin C (MC) and X-rays. The results are best explained if caffiene inhibits a post-replication repair process, particularly the filling-in of gaps in the newly synthesized DNA.     

Pre-replication recovery from induced chromosomal damage

Barley seeds were treated with diethyl sulphate (DES) or methyl methane-sulphonate (MMS) and stored at 30% water content for 3–28 days, i.e. under conditions artificially prolonging the period between the mutagenic treatment and the onset of DNA synthesis and favourable for the repair of induced DNA single-strand breaks. Storage of seed led to a marked decrease in the frequency of induced chromosomal structural changes (aberrant metaphases) as compared with treatments without storage. The recovery involved mainly chromatid-type aberrations, whereas chromosome-type aberrations induced by MMS were recovered to a lesser extent. As no, or only a negligible, DNA synthesis took place in the course of seed storage at 30% water content, the recovery from the induced chromosomal aberrations (more exactly the repair of DNA lesions responsible for aberrations) may probably be ascribed to the pre-replication type of repair. No similar recovery was observed after exposure of seeds to X-rays.     

The nature and repair of DNA lesions that lead to chromosomal aberrations induced by ionizing radiations

Short treatment (up to 1 h) of cytosine arabinoside (araC) increases the frequencies of aberrations induced by X-rays in human lymphocytes, evaluated at the first mitosis following stimulation, or as prematurely condensed chromosomes of G₀ nuclei. Parallel biochemical experiments using nucleoid sedimentation technique, demonstrate that araC inhibits rejoining of DNA-strand breaks effectively. These results point out that X-ray-induced short-lived DNA strand breaks lead to chromosomal aberrations in human lymphocytes.     

Analysis of the visible mutations induced by x-rays and ethyl methanesulfonate in the mature spermatozoa of Drosophila melanogaster

The recessive visible mutations spectrum of chromosome II induced by X-rays and ethylmethanesulfonata (EMS) in mature Drosophila melanogaster spermatozoa has been studied. Treatment of both mutagens resulted in mutations in all 5 genes in stock mei-9LI and only 4--in D-32. The comparison of mutation frequencies of the same genes in two stocks under EMS-treatment demonstrated the statistical difference of mutation frequencies j, pr, cn of two stocks, genes b and vg did not differ. Under the influence of X-rays the differencies have been observed only for gene b. In stock D-32 the mutation frequency differes from the control for b and vg (EMS treatment) and j, pr, vg (under the action of X-rays), in mei-9LI--the mutation frequency of all 5 genes (under the X-rays) and 4 of 5 genes (EMS treatment).