F.H. Sobels

The advantages of drosophila for mutation studies

Summary The advantages Drosophila has to offer for studies on the induction of mutations and chromosome aberrations are in anifold. The wealth of specific tester strains, special markers, useful inversions, or other rearrangements makes it possible to test in one and the same experiment for the total spectrum of genetic changes ranging from dominant lethals and chromosome loss (as indication for non-restituted breaks) to translocations (misrepaired breaks), recessive lethals, deletions, crossing-over, nondisjunction, complete and mosaic visible mutations in a whole array of different germ cell stages. Moreover, a detailed genetic analysis of each of these is possible and thus may shed light on mechanisms and consequences of induction of the genetic changes involved. Studies on the ability of agents to produce both gene mutations and chromosome aberrations are particularly important when evaluating the relevance of current testing procedures for environmental mutagens in mammalian systems that are based on chromosome reakage effects. Thus, hycanthone, diethyl nitrosamine and diethyl sulphate that all produce recessive sex-linked lethals at high frequency in Drosophila, but no chromosome breakage, would erroneously register as safe in most mammalian test systems. New methods for the detection of autosomal non-disjunction have recently been worked out, and deficiencies which are expected to arise with a much higher frequency after chemical treatment than with X-rays, can be easily discovered by employing simple recessive lethal tests. One piece of recent evidence that strengthens the premise of the suitability of Drosophila for studies in chemical mutagenesis, is that in tests of “indirect mutagens” the flies appear to carry out the same activating transformations as those occurring in the mammalian liver. Furthermore, exact methods for chemical dosimetry are available. In addition to all other advantages, Drosophila certainly offers the quickest test system for mutations available in animals today. Consequently, it may provide guidance on the kind of questions that should be looked for in more laborious systems. Thus, observations on modifications and specificities in Drosophila, show at least that a particular treatment can be modified, and this may then be followed up in mammalian cells.

The inhibiting effect of caffeine on the maternal repair of radiation-induced chromosome breaks in drosophila

Summary Drosophila females were treated with caffeine and mated to males that had been exposed to 2000 R X-irradiation; only mature spermatozoa were sampled. The frequencies of radiation-induced dominant lethals, sex-chromosome losses, and II-III translocations in the paternal genome were determined. Treatment of the females with caffeine led to an increase in the frequency of dominant lethals and sex-chromosome loss and to a decrease in the frequency of translocations. If it is assumed that dominant lethals and sex-chromosome loss arise mainly from open, non-restituted breaks, and that translocations are the result of misrepaired breaks, the present findings suggest that caffeine acts by partially inhibiting repair processes acting in the oocytes, processes that are required for the repair or misrepair of chromosome breaks induced in the spermatozoa.

The role of oxygen in determining initial radiosensitivity and post-radiation recovery in the successive stages of Drosophila spermatogenesis

Abstract The role of oxygen in determining radiosensitivity to the induction of premutational damage, and in processes of post-radiation repair has been studied in different stages of spermatogenesis by comparing the effect of post-treatment with N2 to that with O2 after X-irradiation of Drosophila males in either N2 or O2. Recessive lethals were scored in a ring-X chromosome in 4–5 successive two-day broods. After irradiation in N2, it is found that post-radiation recovery in sperm is favoured by N2, but in early spermatids and spermatocytes by O2. In late spermatids and spermatogonia no significant post-radiation modifications have been observed. Post-radiation recovery by N2 in mature sperm has been further confirmed by results obtained from sperm, treated in the inseminated females. This effect is most pronounced in sperm, derived from the first mating that is used for fertilizations during the first three days after irradiation. It is pointed out that the observed post-radiation modifications cannot be explained by shifts in the sampling of germ cells with different radiosensitivities between flies having received different post-treatments, nor by selective elimination of germ cells with mutations. The results for early spermatids and spermatocytes indicate that a repair system which has been rendered ineffective by anoxia can be regenerated to normal functioning by post-treatment with O2. The time of mutation fixation in early spermatids could not be established definitely, but indications were obtained that in N2 fixation does not occur within 25 min, whereas after 50 min the reversability of the pre-mutational lesions is strongly decreased. In contrast to the observations after irradiation under anoxia, no post-radiation modification of the mutation frequencies by N2 or O2 is found after irradiation in O2 for sperm, spermatids or spermatocytes. Only in spermatogonia post-treatment with O2 lowered the mutation frequency, as compared to that after N2 post-treatment. It is concluded that radiation in O2 produces more irreparable damage than radiation in N2. By comparing the mutation frequencies after irradiation in O2 with those obtained in N2, under conditions of inhibited repair, that is, for O2 post-treatment in sperm, and for N2 post-treatment in early spermatids and spermatocytes, oxygen enhancement ratios for initial radiosensitivity to the induction of pre-mutational damage could be determined; these are about 1.6 for sperm, 3.3 for spermatids, 2.4 for spermatocytes and 3.8 for gonia. O2-dependent radiosensitivity in spermatids thus is considerably greater than in sperm. On the basis of the above observations, when considered together, the differences in response to radiation in air between spermatozoa and early spermatics can now be satisfactorily explained. Also an interpretation is possible, why in earlier reports9, 10, 22–24, 29, 33 radiation in N2, when followed by air, levelled off the difference in sensitivity between sperm and spermatids, but higher yields of mutation were invariably obtained from spermatids than from sperm after irradiation in O2 (ref. 5).

Models and assumptions underlying genetic risk assessment

Various methods employed for estimating the genetic risks of radiation are reviewed. With the doubling-dose method, genetic damage is expressed as an increase in cases of known genetic disease. The actual doubling dose is based on figures obtained with the mouse. There have been no recent data on induced mutation frequencies. Recent results suggest that the prevalence figure for multifactorial disease may be at least one order of magnitude higher than before. Various assumptions underlying the doubling-dose concept are discussed in the light of recent findings on: (1) spontaneous mutations resulting from insertion elements, and (2) the comparability between spontaneous and induced mutations. The so-called direct method makes use of figures for induction of dominant mutations affecting the skeleton and the lens of the eye in the mouse, and of translocation induction in monkeys. Induction rates are converted to overall rates of induced dominant effects in man by applying certain assumptions. The proportionality between dose and effect is the basis for all genetic risk assessments. The possible significance of data on human lymphocytes indicating a threshold below 4 rad and the induction of repair enzymes by low radiation doses is discussed. The parallelogram approach is based on the principle that estimates can be obtained on the amount of genetic damage that cannot always be assessed directly. Thus mutations in mouse germ cells can be predicted by using mutation frequencies in cultured mammalian cells and O6-ethylguanine adducts. Measurement of haemoglobin mutations in human and mouse erythrocytes, and of HPRT-deficient mutations in lymphocytes of man and mouse should make more precise estimates of mutation frequencies in human germ cells possible. The development of a database on mutations in somatic cells of the mouse, their induction frequencies and molecular nature are considered an important priority. Used in combination with mouse germ-cell mutation frequencies, they should enable more precise risk estimates on the basis of mutations in somatic cells of man.

The effects of treating drosophila females with actinomycin-D on the yields of dominant lethals, translocations and recessive lethals recovered from X-irradiated spermatozoa

Abstract Drosophila females were injected with Actinomycin-D. After a day they were mated (for 24 h only) to males that had been exposed to 2000 or 2500 R X-irradiation, so that only mature spermatozoa were sampled. The radiation-induced frequencies of dominant lethals, II–III translocations and recessive sex-linked lethals in the paternal genome were determined. When compared to the appropriate controls, it is found that such treatment of the females with Actinomycin leads to an inrease of the frequency of dominant lethals and to a decrease of those of translocations and recessive lethals. Characteristic is the delayed expression of the Actinomycin effect, in that the modification of translocation and recessive lethal frequencies is most pronounced in oocyte stages utilized 4–6 days after injection. The present findings are interpreted to mean that Actinomycin acts by partially inhibiting the restitution of chromosome breaks, thereby increasing the frequency of dominant lethals, and decreasing that translocations and recessive lethals. This implies that maternal repair processes acting at the stage of pronucleus formation are required for the repair (restitution) or misrepair (reunion giving rise to translocations) of chromosome breaks induce mature spermatozoa.

The effects of sodium fluoride and iodoacetamide on mutation induction by x-irradiation in mature spermatozoa of Drosophila.

Abstract The effect of two inhibitors of glycolysis, NaF and iodoacetamide on the production of recessive lethal mutations by X-rays in mature Drosophila sperm has been investigated. Pre-treatment with NaF resulted in a consistent and highly significant increase of the mutation frequency. This effect is thought to result from interference with a repair process which makes use of energy produced by glycolysis, and does not arise from an increase of the oxygen tension. When the action of NaF was studied in combination with pre- and post-treatments with N 2 and O 2 , it was observed: (1) that irrespective of pre-treatment with N 2 or O 2 , NaF enhanced the mutation frequency over that in the saline controls; (2) that following irradiation under anoxia, post-treatment with N 2 reduced the mutation frequency below that observed with O 2 post-treatment, even when the flies had been pre-treated with NaF. These additive effects of NaF pre-treatment and O 2 post-treatment have been taken as an indication that, even when glycolysis is inhibited by NaF, some energy is left, which is still available for repair by post-radiation anoxia. This interpretation that the amount of repair in sperm depends on different levels of available energy is supported by the observation that NaF pre-treatment is still effective in increasing the mutation frequency over that in the controls, when N 2 was given before, during and after irradiation. Thus repair is maximal with NaClN 2 RN 2 , intermediate with NaFN 2 R 2 N 2 , and minimal with NaFN 2 RO 2 . Pre-treatment with iodoacetamide gave more variable results than that with NaF.

Induction by x-rays of isochrosomes in the germ cells of Drosophila melanogaster males evidence for nuclear selection in embryogenesis

Abstract Drosophila melanogaster males, heterozygous for markers on either the second or third chromosomes, were irradiated and mated to females in which the corresponding homologous pair were present as isochromosomes. Gametes at different stages of development at irradiation were sampled by serially mating the males in 5 2-day broods. All normal zygotes were inviable; most of the viable progeny carried newly induced isochromosomes or were triploid. The new isochromosomes were either heterozygous or homozygous for the paternal markers. The former, heteroisos, were only induced in diploid cells and the latter, homoisos, were induced in all of the germ-cell stages which were tested. The rates of induction could not be measured directly but were estimated from the results of some calibration tests. Spermatocytes and spermatogonia appear to be about 30 times more sensitive than spermatozoa and mature spermatids for the induction of homoisos. The unexpected finding is that homoisos, which presumably result from a chromatid-type exchange, can be recovered from irradiated spermatozoa and late spermatids. The survival of such induced homoisos is explained by taking into account the process of gametic fusion of Drosophila. Only one of the first two fusion nuclei will be genetically balanced and competent to produce an adult. Triploids resulted from the production of diploid female gametes. The data provide a strong indication that this is not a random process.

A study of the causes underlying the differences in radiosensitivity between mature spermatozoa and late spermatids in drosophila

Abstract The basis for the differences in radiosensitivity between mature spermatozoa and late spermatids was analysed by determining recessive lethal and translocation frequencies following irradiation in O 2 , N 2 and air. Spermatozoa were sampled from the first ejaculate of 7-day-old males and spermatids from that of I-h-old males. The results indicate that the higher sensitivity of spermatozoa than of late spermatids after irradiation in air originates from a higher degree of oxygenation in the former than in the latter cells. Spermatozoa are not characterized by a greater sensitivity to irradiation in the presence of O 2 because the same oxygen enhancement ratios were recorded for both types of cells. These findings indicate that in late spermatids, a mechanism other than the one postulated to explain differential sensitivity of the early spermatids (which are characterized by an oxygen enhancement ratio of 3.3 and thus by greater radiosensitivity in O 2 than spermatozoa) operates. The somewhat greater sensitivity to irradiation in air of spermatozoa in the male than in the inseminated female is also ascribed to a higher degree of oxygenation, because equal yields of recessive lethals were obtained after irradiation in both O 2 or N 2 . After radiation exposure in O 2 higher translocation yeilds were obtained from spermatozoa in females than in males. Since the lethal data suggest that the level of genetic radiation damage induced is approximately the same in both kinds of sperm, it is inferred that the conditions for repair of lesions leading to aberrations are less favourable when sperm is exposed to irradiation in O 2 in females than in males. In confirmation of an earlier report by Falk , mating of irradiated males to females with an attached X · Y-chromosome resulted in an increased recovery of II-III translocations.

The capacity of Drosophila for detecting relevant genetic damage

For the detection and study of mutagenic agents, Drosophila offers many advantages. It is a higher organism with a short generation time that is cheap and easy to breed in large numbers. The simple genetic testing methods provide unequivocal answers about the whole spectrum of relevant genetic damage. A comparison of the detection capacity of assays sampling different kinds of genetic damage revealed that various substances are highly effective in inducing mutations, but do not produce chromosome breakage effects at all, or only at much higher concentrations than those required for mutation induction. Of the different assay systems available, the classical sex-linked recessive lethal test thus deserves priority, in view of its superior capacity to detect mutagens. Of practical importance is also its high sensitivity, because a large number of loci in one-fifth of the genome is tested for newly induced forward mutations, including small deletions. Drosophila is capable of carrying out the same metabolic activation reactions as the mammalian liver. An additional advantage, in this respect, is the capacity of Drosophila for detecting short-lived activation products, because intracellular activation occurs within the spermatids ans spermatocytes. These properties make the test for recessive sex-linked lethals a useful tool for verifying results obtained in the pre-screening of potential mutagens with fast microbial assay systems. In studies on non-disjunction, detailed genetic analysis of the induced changes is possible, and these may shed light on the mechanisms involved. A new adaptation of the bithorax transvection method by Mendelson permits the recovery of high yields of chromosome aberrations in a fast one-generation test.

The persistence of chromosome breaks in different stages of spermatogenesis of Drosophila.

Abstract To determine whether chromosome breaks produced in spermatids and spermatocytes remain open until after fertilization, fractionation experiments with pupae of different ages were carried out. In a first series of experiments the first fraction of 500 R X-irradiation was given to 0-, 24-, or 48-h-old pupae, and the second fraction of 1000 R to inseminated females, i.e. to the same cells after they had developed into mature spermatozoa. Simultaneously, appropriate control experiments with irradiation of the pupae or inseminated females alone were carried out. The translocation yield obtained in the fractionated group was then compared with that expected on the basis of additivity or interaction between breaks produced by the single fractions. The results show that ( i ) in the o-h pupal series, the translocation yields fall somewhat short of additivity; ( ii ) in the 24-h series, the frequency o did not deviate from additivity; ( iii ) in the 48-h series there was good evidence for interaction of breaks. In a second set of experiments, the pupae received either ( i ) a total exposure of 800 R delivered in two fractions, the first one when they were 24 or 36 h old, and the second when they were, respectively, 32 or 45 h old; or ( ii ) a single exposure of 400 R when 24, 32, 36 or 45 h old. From the yield of translocations obtained with the latter regime, the frequencies on the basis of additivity (and interaction) were computer and compared with those actually observed with regime ( i ). It was found that ( i ) when the first fraction is given to 24-h-old pupae, the translocation yields are entirely consistent with the expectation based on additivity; ( ii ) when the first fraction was given to 36-h-old pupae, the yield conformed to interaction. Transition from additivity to interaction of breaks thus occurs between 24 and 36 h of pupal age. A comparison of the translocation frequencies induced by single 400 R exposures in pupae of different ages, from cultures with controlled population density, revealed that spermatids in 24-h-old pupae are characterized by peak sensitivity to X-irradiation.