Irradiation of male rats increases the chromosomal sensitivity of progeny to genotoxic agents
I.E.Vorobtsova
Laboratory of Radiation Genetics, Central Research Institute of Radiology and Roentgenology, St.Petersburg, Pesochny-2, Leningradskaya, 70/4, 189646, Russia.
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Abstract
The chromosomal sensitivity to genotoxic agents was studied in the first generation progeny of male rats irradiated at the dose of 4,5 Gy of X-rays and in the progeny of nonexposed animals. The frequency of anaphase chromosome aberrations (bridges or/and fragments) in rats exposed to X-rays or treated by cyclophosphamide was estimated: in the proliferating hepatocytes (2 Gy) as a function of time during liver regeneration after partial hepatectomy; in bone marrow cells (2,5 Gy or 25 mg/kg body weight) and in fetal fibroblasts (3 Gy). The sensitivity of chromosomes to genotoxic agents was found to be increased in the progeny of irradiated male rats as compared to the progeny of nonexposed animals. This finding provides supportive evidence that irradiation of parents is an important factor of predispositionof progeny to chromosomal instability.
Introduction
Among the genetic effects of ionizing radiation in mammals are severe disorders in the progeny of irradiated parents or so called ”untoward pregnancy outcomes”: fetal and early postnatal death, malformations, sterility, congenital abnormalities (both morphological and biochemical) and hereditary diseases (UNSCEAR 1986). Although found in rodents and other animals, such genetic consequences of radiation have not so far been identified in humans (Neel et al, 1990). It has been proposed (Vorobtsova, 1974; 1989) that the frequency of these genetic effects depends on the rate of reproduction of the species concerned. The higher this rate the more tolerant the species will be to defective offspring, and the more likely will express severe consequences of parental irradiation.
In the 50’s and 60’s several studies were performed (mainly on Drosophila and rodents) to investigate whether the phenotypically normal progeny of irradiated animals show normal fitness (Wallace, 1958; Spalding et al, 1963; Mukai et al, 1966; Falk, 1967; Toropanova, 1962; Shilenko, 1965; Russell, 1957; Newcomb and McGregor, 1964; Sheridan and Ronnback, 1967; Zemtsova and Osypovski, 1960). The data obtained have been rather contradictory and difficult to compare because of variations in experimental methods, the number of generations studied and the end points used. In our previous studies of fitness of phenotypically normal offspring of irradiated animals we carried out experiments only with the first generation progeny of irradiated males, we estimated simultaneously several end points in cells, tissues and at the organism level, and used various challenges to reveal the possible deficiencies in progeny which would not be evident without such challenges (Vorobtsova et al, 1967; Vorobtsova and Safronova, 1967; Vorobtsova and Jurieva, 1972; Vorobtsova, 1974, 1978, 1989; Vorobtsova and Golsberg, 1982; Fokina and Vorobtsova, 1987). These results obtained in Drosophila and rodents clearly demonstrated the existence of radiation-induced genetic effects manifested in impaired fitness in progeny of irradiated animals. The expression of these effects depended strongly on the genetic background of the strain studied, on the nature of the challenge, and the type of end point used.
An increased cancer risk as a genetic effect of parental irradiation has been demonstrated in rodents (Novikova, 1966; Pronina, 1968; Alexandrov et al, 1974; Streltsova et al, 1982; Nomura, 1983; Vorobtsova and Kitaev, 1988; Vorobtsova et al, 1993). These results are in good agreement with the animal data available on chemically induced multigeneration carcinogenesis (Nomura,1978; Tomatis, 1989; Napalkov et al, 1987).
It is well known that both cancer itself and a hereditary predisposition to tumors are usually accompanied by genetic (chromosomal) instability (Hagmar et al, 1994; Cloose et al, 1994; West et al, 1995). Some years ago we proposed that paternal irradiation can lead to the chromosomal instability in the progeny (Alexandrov et al, 1974) and we obtained preliminary results supporting this suggestion on hepatocytes of the progeny of irradiated male rats. Later on we and others were able to confirm these data in other systems (Vorobtsova, 1987, 1989; Fokina and Vorobtsova, 1987; Vorobtsova et al, 1993; Luke et al,1997).
Here we report on the chromosomal instability of the progeny of irradiated rats assessed in various somatic cells using various mutagenic challenges.
Materials and methods
Animals
Outbred white rats from the “Rappolovo” animal farm of the Russian Academy of Medical Sciences were used. The animals received standard laboratory chow (Baranova et al, 1986) and tap water ad libitum. A temperature of 20-22oC and a cycle of 12 hours light/12 hours dark was maintained in the room.
Irradiation and experimental design
For whole-body X-irradiation male rats were placed in Plexiglas boxes surrounded by paraffin phantoms on all sides except the top. Irradiation of animals at the dose 4,5 Gy was performed using a RUM-3 X-ray machine (USSR) operated at 220 kV and 15 mA, with 0.5 mm Cu and l mm Al filters. The dose rate was 0.80.02 Gy/min. After irradiation each male was individually mated with two nonexposed females over a period of 3 to 4 days and then removed. After weaning offspring’s were kept 5-6 animals per cage, males and females separately. The offspring of control animals were kept under the same conditions. Cytogenetic effects of radiation and chemicals were studied in hepatocytes, bone marrow cells and fetal fibroblasts in the progeny of nonirradiated (F1c) and irradiated (F1i) animals of both sex.
Experiments with hepatocytes
Both F1i and F1c rats were exposed to X-rays (2 Gy) at the age of 6 months. After irradiation, partial hepatectomy was performed to stimulate cell division. Animals were killed at 24, 30, 33, 36, 39, 42 and 48 hours after operation to compare the kinetics of the cytogenetic effects of radiation between F1i with F1c during regeneration of liver. Pieces of regenerating liver lobes were fixed in ethanol/acetic acid (3:1) and stained in Feulgen reagent. Small fragments of liver tissue were placed on slides in a drop of 45% acetic acid, mounted under cover slips and squashed. Slides were put on the surface of dry ice for several minutes. Then after removing of coverslips slides were dehydrated with 70%,90%,96% alcohol, treated by xylene and mounted in Canadian balsam.
Experiments with bone marrow cells
The F1iand F1c animals were subdivided into 3 groups. Group 1 animals were irradiated at the age of 3 months (dose - 2.5 Gy) and killed 8, 16 and 24 hours after irradiation. The rats of group 2 were injected with an aqueous solution of cyclophosphamide (25 mg per kg body weight) at the age range of 3-11 months and killed 24 hours later. Animals in group 3 received no treatment. The bone marrow was fixed in femurs by ethanol/acetic acid (3:1), stained by Feulgen reagent, and the slides from pieces of bone marrow were prepared as described (see Experiments with hepatocytes).
Experiments with fetal fibroblasts
Female rats mated with either irradiated or nonirradiated males were killed under ether narcosis on the 18th or 19th day of pregnancy. The embryos were removed from the uterus and fetal fibroblast cultures were set up in Carrel flasks with Eagle medium; the seeding concentration was 8 x 105 cells/ml. The growth of cells was checked under a phase-contrast microscope. Cells reached a confluent monolayer (usually on the 3rd-4th days after seeding) were removed from the bottom of flask by 0,02% chemotrypsin treatment, carefully resuspended in fresh culture medium and put into two fresh Carrel flasks at the same seeding concentration. The following passages were produced by the same way. Irradiation of fibroblasts reached confluent monolayer (dose - 3 Gy) was carried out in 0,1,2,3,4,5,6 and 7 successive passages. After irradiation fibroblasts were harvested and reseeded (8x105 cells/ml) in tubes with narrow coverslips inside. After 48 hours of cultivation the coverslips with attached cells were removed from the tubes, washed in phosphate buffer, fixed in ethanol/acetic acid (3:1) and stained by Giemsa. In each passages the same procedure was performed with nonirradiated fibroblasts.
Analysis of chromosome aberrations
Chromosome aberrations were studied by anaphase method .Coded slides were analyzed under light microscope with oil immersion objective (x900). At least 100 cells of late anaphase-early telophase were scored for each animal or each passage of fibroblasts and the total number of aberrations per 100 cells was calculated. Two types of chromosome aberrations were registrated - bridges which are known to arise from asymmetrical chromosomal exchanges and fragments. Bridge with a fragment was registrated as one aberration, excess fragments - as separate mutation events.
Statistics
Computer statistical analysis was made using Chi-squared and variance ratio F-tests. A significance level of 0.05 was used throughout.
Results
The dose of 4,5 Gy did not influence survival and mating ability of male rats during mating period: 3-4 days after irradiation and reduced litter size from 9,9±0,5 (control) to 6,8±0,6 offsprings per female. It means that this dose induced about 30 % of dominant lethals and the number of nonlethal damages in parental germ cells could be enough to reveal their effect on the chromosomal stability of progeny. Moreover this dose of parental exposure affect the fitness of progeny as it was found in our previous studies (Vorobtsova, 1989).
In Table 1 the results are presented on chromosome aberration frequency in hepatocytes at various times after irradiation of rats with a dose of 2 Gy and partial hepatectomy. The aim of this experiment was to ascertain whether the increased radiosensitivity of F1i hepatocytes reported earlier (Vorobtsova, 1987, 1989) is an intrinsic property of the cells and could not be attributed to changes in population structure during the process of cell proliferation.
Through the whole period studied the number of chromosome aberrations in hepatocytes of F1i was significantly increased as compared to F1c. Spontaneous level of chromosome aberrations in hepatocytes did not differ significantly in groups compared, being 7,5±0,9 in F1i and 7,2±0,3 in F1c (Vorobtsova, 1987) The frequency of aberrations in both groups was the lowest at 24 hours after partial hepatectomy. It is worth noting that the number of damages per aberrant cell was also less at this time then at the period from 30 till 48 hours after operation. For F1c these values were 1,440,11 and 1,810,06; for F1i - 1,540,03 and 1,920,06 respectively. This decreased aberration frequency at the earliest time of fixation of proliferating hepatocytes appears to be due to the delayed progress of aberrant cells through mitosis.
The data obtained in experiments with bone marrow cells are presented in Table 2. The spontaneous number of chromosome aberrations per 100 cells varied from 4,8 to 9,8 in F1c and from 4,0 to 11,2 in F1i and mean aberration yields did not differ significantly between groups compared (F-test). The reason of such a variability of spontaneous aberration frequency as well as high values in some sets of experiment is not clear. Since the majority of aberrations observed in karyocytes of nontreated animals were fragments some uncontrolled factors (for example virus infection) could be involved. To study the chromosomal sensitivity to cyclophosphamide the dose of 25 mg per kg body weight was used. Mutagenic effect of this dose was clear enough but not too high to wipe off possible difference between groups. The frequency of chromosome aberrations in karyocytes of rats treated with cyclophosphamide at various ages and killed 24 hours later when maximal cytogenetic effect of this substance takes place (Datta and Schleiermacher, 1969) was significantly higher in F1i as compared to F1c. The distribution of rats treated with cyclophosphamide and showing a given number of aberrations corresponded to the binomial law (2 test). It was shifted to the right in F1i as compared to F1c (Figure 1), as seen previously with hepatocytes (Vorobtsova,1987).
The results of study of bone marrow cell radiosensitivity are presented in Table 3.The frequency of induced aberrations in both groups is highest at the 8 hour after irradiation and then falls. One of the possible explanation of this observation is that at this time aberrations were scored in anaphase cells being at the time of irradiation in the G2 phase of the cell cycle which is known to be the most sensitive one. The frequency of radiation induced damages in F1i was higher than in F1c during whole period of investigation. The number of aberrations in karyocytes of nonexposed rats of the same age (3 months) is higher in F1c than in F1i group (see Table 2) but not significant (F-test).
The results of experiments with cultured fetal fibroblasts are presented in Table 4. One can see that there are no systematic changes in the number of aberrations, through the passages both for irradiated and non-irradiated cultures of F1i and F1c. The frequency of chromosome aberrations in irradiated cultures is significantly higher in the F1i progeny as compared to the F1c one. As far as spontaneous level of aberrations does not differ significantly in these groups (F-test) it gives reason to propose the increased radiosensitivity of fibroblasts in the progeny of irradiated male rats.
Thus on three types of somatic cells (hepatocytes, bone marrow karyocytes and cultivated fetal fibroblasts) the chromosomal sensitivity to genotoxic agents in the cells of progeny of irradiated male rats was found to be increased as compared to the progeny of nonirradiated parents.
Discussion
Genetic instability observed in some human hereditary syndromes (xeroderma pigmentosum, ataxia telangiectasia, Fanconi’s anemia and others) is accompanied by an elevated rate of spontaneous chromosomal aberrations, hypersensitivity to mutagens and increased cancer risk (Cleaver, 1970; Epstein et al, 1973; Akifiev et al, 1983; McKinnon, 1987). For xeroderma pigmentosum these effects are known to be due to mutations in the genes responsible for DNA repair. There is in fact a lot of evidences from different organisms (plants, Drosophila, fishes and humans) clearly demonstrating that not only single gene mutations, but random structural (Lucchesi, 1986) and numerical rearrangements of karyotypes (Akifyev et al, 1983) as well as artificial genome manipulations such as inbreeding (Gorbunova and Kaydanov, 1975), crossbreeding (Belgovsky, 1937), transfection of DNA (Nabirochkin et al, 1987) are able to modify spontaneous mutation rate.
In the 1950s and 60s studies with various cells (bacteria, yeast and mammalian cells in vitro) were performed clearly demonstrating that irradiation could cause not only early effects but also delayed ones. These late effects were manifested in increased death and mutation rates in many generations of the progeny of irradiated cells (Puck and Marcus, 1956; Sinclair, 1964; Beer, 1979; Bychkovskaya and Ochinskaya, 1977; Bychkovskaya, 1986; Seymour et al, 1986). Similar delayed chromosomal effects have been reported more recently in irradiated cells (Zloba and Sevankajev, 1991; Chang and Little, 1992; Holmberg et al, 1993; Kadhim et al, 1995). All these data provide supportive evidence that irradiated cells have a “long term memory“ which is expressed in genetic instability some time later. This “memory“ can also be manifested as hypersensitivity of the progeny of irradiated cells to mutagenic challenge. Data obtained using bothin vivo andin vitro assays showed that irradiation at moderate doses can induce hypersensitivity to mutagenic factors which are applied long after the initial exposure to radiation. Prior irradiation of mice at the dose-range 0.12-3 Gy increases the radiosensitivity of G2 bone marrow chromosomes (Jacobson-Kram and Williams, 1988). Increased chromosomal radiosensitivity of lymphocytes is characteristic of patients who have undergone radioimmunoglobulin therapy (Xiao et al, 1989). The hypersensitivity of X-irradiated V79 cells to PUVA treatment was found to persist for more that 200 generations (Frank and Williams, 1982). A similar effect has been demonstrated on irradiated HeLa cells (Taponinen et al, 1986; Pelevina et al, 1993; Chernikova et al, 1993).
Hypersensitivity can be induced by other mutagens as well. V79 cells irradiated with medium wavelength UV had an increased yield of HPRT mutations after UV challenge compared to nonirradiated cells exposed to UV (Ikebuchi et al, 1988; D’Arpa et al, 1989). The cytogenetic response of lymphocytes to in vitro treatment with pesticides was found to be increased in people occupationally exposed to these chemicals (Pilinskaya, 1985).
In all the studies mentioned the instability (and/or hypersensitivity to mutagens) was observed in the mitotic descendants of irradiated cells. Our previous studies support the conclusions of present study in that this effect is observed in the cells of the sexual F1 progeny of irradiated parents (Alexandrov et al, 1974; Vorobtsova, 1987, 1989). In present study these results were confirmed by experiments where the frequency of aberrations in F1i and F1c was followed with time in regenerating liver after partial hepatectomy as well as in experiments performed on the other types of somatic cells (bone marrow karyocytes and fibroblasts) and with another mutagenic challenge (cyclophosphamide). Recently a 2 fold increase in spontaneous mutability was demonstrated in haemopoetic cells of F1 generation mice of irradiated parents (Luke et all,1998).
The phenomenological resemblance of these somatic and genetic effects of ionizing radiation described suggests that the mechanisms of genomic instability in both cases could be similar. The mass character of alterations in the progeny of irradiated parents (see Figure 1) and the variety of cells affected provide evidence that such a mechanism is unlikely to involve unique gene mutations because of their rarity (UNSCEAR, 1986). The effect presumably could be due to mutations in hypervariable minisatellite loci. Recently it has been shown that such mutations occur much more frequently then specific locus mutations (Jeffreys, 1987; Dubrova et al, 1993). It is worth noting that the important role of mutations of polygenes controlling viability in nonspecific genetic effects of radiation (decreased fitness) was proposed some time ago (Mukai et al, 1966). Another (but not alternative) possibility to explain the instability of irradiated parents progeny is that some epigenomic events are involved (Alexandrov, 1982). Among them could be for example radiation-induced changes in DNA-protein interaction which make chromosomes more mutable; these changes can persist during both mitotic and meiotic cell division. Recent data provide supportive evidence in favor of this point of view (Schwartz and Vaughan, 1993). In this study of DNA-nuclear matrix interactions both in radioresistant and radiosensitive human cell lines, an association was found between inherent radiation sensitivity and the ability of DNA supercoiled loops to relax and unwind in some experimental conditions. Our previous data (Vorobtsova, 1974; 1989), showed that in the progeny of irradiated animals many characteristics of their fitness are changed, also support the proposal that the target of radiation-induced instability is of polygenic and/or epigenomic nature. The discovery of a genetic effect of ionizing radiation manifested in the form of chromosomal instability should it seems be taken into account when assessing risks and possible health detriment for offspring of exposed parents. Earlier studies (Alexandrov et al, 1974; Nomura, 1982; Vorobtsova, Kitaev, 1988; Vorobtsova et al, 1993) as well as recent publications (Lord et al, 1998) clearly demonstrated one of such a health effect of preconceptional parental irradiation - an increased cancer risk in the progeny.