|Year : 2018 | Volume
| Issue : 2 | Page : 79-85
The comparison of genetic instability in haploid and diploid yeast cells exposed to ionizing radiations of different linear energy transfer and ultraviolet light
Ekaterina S Evstratova1, Kaushala P Mishra2, Vladislav G Petin1
1 A. Tsyb Medical Radiological Research Centre, Branch of the National Medical Research Radiological Centre, Ministry of Health of the n Federation, Obninsk 249036, Russia
2 Bhabha Atomic Research Center, Mumbai, Maharashtra, India
|Date of Web Publication||22-May-2018|
Dr. Kaushala P Mishra
Bhabha Atomic Research Center, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Context: It is analyzed whether genetic instability is determined by cell ability to recover from radiation damage or it is mainly determined by cell ploidy. The values of the RBE of α-particles for cell survival and genetic instability are obtained for haploid and diploid yeast cells. The delayed appearance of clones by cells surviving after exposure to γ-rays, α-particles, and UV-light are compared. Aim: To compare of genetic instability in haploid and diploid yeast cells exposed to ionizing radiations of different LET and UV light. Materials and Methods: Haploid (strain S288C, RAD) and homozygous diploid (strain XS800, RAD/RAD) yeast cells of Saccharomyces cerevisiae were used in our experiments. Cell survival and delayed clone appearance were studied for cells surviving after exposure to 60Co γ-rays, 239Pu α-particles and 254 nm UV light. Survival was determined by cell ability to produce macrocolonies on a solid nutrient medium. Genetic instability was defined by the delayed appearance of clones by cells surviving irradiation. Results: The delayed appearance of clones by cells surviving after irradiation has been well expressed and reached about 100% for diploid strain and only 20–25% for haploid strain independently of radiation type. Both cell survival and genetic instability exhibited more pronounced manifestation after the action of alpha particles than after irradiation with gamma-rays. This effect may be associated with greater efficiency of densely ionizing radiation to produce lethal radiation damage and accompanying sub-lesions responsible for delayed appearance of clones. The dependence of this effect on cell survival was substantially the same after exposure to UV light, sparsely and densely ionizing radiation. Conclusion: The genetic instability is mainly determined by cell ploidy rather than the shape of survival curve and the ability of cell to recover from radiation damage as it is traditionally assumed for Saccharomyces cerevisiae yeast cells.
Keywords: Alpha particles, gamma rays, genetic instability, survival, ultraviolet light, yeast cells
|How to cite this article:|
Evstratova ES, Mishra KP, Petin VG. The comparison of genetic instability in haploid and diploid yeast cells exposed to ionizing radiations of different linear energy transfer and ultraviolet light. J Radiat Cancer Res 2018;9:79-85
|How to cite this URL:|
Evstratova ES, Mishra KP, Petin VG. The comparison of genetic instability in haploid and diploid yeast cells exposed to ionizing radiations of different linear energy transfer and ultraviolet light. J Radiat Cancer Res [serial online] 2018 [cited 2021 May 6];9:79-85. Available from: https://www.journalrcr.org/text.asp?2018/9/2/79/232985
| Introduction|| |
Genetic instability concerns such important phenomena as radiation mutagenesis, carcinogenesis, and aging, the main long-term effects of ionizing radiation and ultraviolet (UV) light., Genetic instability refers to chromosomal aberrations, reproductive cell death, genome rearrangement, malignant transformation, reduced cloning efficiency, micronucleus formation and heterogeneity among the progeny of irradiated cells. The delayed appearance of clones by cells surviving exposure to ionizing radiation can be also considered as an example of genetic instability of irradiated cell.,, The common property of various delayed effects of radiation is the transmission of sublesions to distant progeny. The state of genome instability often precedes tumor progression  and persists a long time after irradiation over many cell generations.,, A poorly studied aspect of this problem is quantitative comparison of survival and genetic instability for haploid and diploid cells surviving after exposure to carcinogenic factors – γ-rays, α-particles, and UV light. The most convenient model for this purpose is yeast cell.,
Densely ionizing radiation with high linear energy transfer (LET) seems promising as cancer treatment because of its higher relative biological effectiveness (RBE) in low-dose region usually used at fractionated irradiation of malignant tumors and due to the suppression of cell recovery from sublethal and potentially lethal damage.,,,, Although the genetic instability is well known after exposure to high LET radiation,,, RBE of densely ionizing radiation has never been quantified for this effect. Moreover, nobody tried to associate the RBE of densely ionizing particles for both cell inactivation and genetic instability. Therefore, the comparison of RBE for both lethal effects and delayed appearance of clones by haploid and homozygous diploid cells remains completely unexplored.
Most results on genetic instability were obtained with cultured mammalian cells in a diploid state. Yeast cells, as the simplest example of eukaryotic cells, are a convenient object for comparing lethal effect and genetic instability of haploid and diploid cells that allows obtaining new fundamental information. In particular, it was shown ,, that the effect of delayed colony formation in yeast cells surviving after irradiation is more expressed for diploid than for haploid cells. There is evidence that DNA double-strand breaks (DSBs) initiate the phenotype of delayed reproductive death. It is well known that DNA DSBs repair requires two homologous chromosomes,, and due to this ability, diploid yeast cells are more resistant to ionizing radiation than haploids whose ability to repair DSBs is greatly reduced and may be realized through nonhomologous end joining., This implies that radioresistant diploid yeast cells showing sigmoid dose-effect curves inherit a number of nonlethal sublesions responsible for genetic instability. Such an effect was found reduced for wild-type haploid yeast cells showing exponential dose-effect curves. On this basis, it was concluded that the shape of survival curve and cell's ability to DNA DSBs repair determine the delayed appearance of clones. However, in our recent work with haploid and diploid yeast cells of wild-type and their radiosensitive mutants, it was noticed  that genetic instability defined by the delayed appearance of clones was well expressed and reached 100% for diploid cells irrespective of the shape of survival curve and cellular ability to recover from radiation damage. This effect was poorly expressed (20%–25%) for all haploid strains characterized by an exponential survival curve. On this basis, a preliminary inference was drawn that the genetic instability induced by ionizing radiation is largely determined by cell ploidy, rather than by the shape of survival curve and cell ability to recover from radiation damage. This assumption, however, needs additional studies for confirmation. It has long been known that the shape of survival curves after irradiation with UV light, unlike the effect of ionizing radiation, does not depend on ploidy and has a sigmoid form for both haploid and diploid yeast cells. Then, a comparison of genetic instability of haploid and diploid yeast cells surviving after UV light exposure could contribute to a better understanding whether this effect is actually related with the shape of survival curve or to a greater extent is determined by the cell ploidy.
In view of above, the present work was aimed to investigate the following: (i) to determine whether genetic instability is determined by cell ability to recover from radiation damage and the shape of the survival curve, or to a greater extent depends on the cell ploidy; (ii) to quantify the RBE of α-particles for cell survival and genetic instability for haploid and diploid yeast cells; and (iii) to compare the delayed appearance of clones by cells surviving after exposure to γ-rays, α-particles, and UV light.
| Material and Methods|| |
Strains and cultivation conditions
Haploid (strain S288C, RAD) and homozygous diploid (strain XS800, RAD/RAD) yeast cells of Saccharomyces cerevisiae were used in our experiments. Yeast cell has a number of properties that make it possible to study the mechanisms of various processes at the molecular, subcellular, cellular, and population levels without using complex technologies and to obtain results in a relatively shorter time period. The short life cycle of yeast and the possibility of rapidly obtaining a large number of cell generation permits to study even certain rare phenomena. Most radiobiological responses of yeast cells, such as survival curve shape, dependence of RBE on LET, oxygen effect, action of radioprotectors, and radiosensitizers, are qualitatively similar to those of mammalian cells.,, Convenience of yeast cell cultivation provides a clear reproducibility of results obtained. In addition, they have other positive qualities: rapid growth on a dense nutrient medium with a large yield of cells; the ability to form homogeneous suspensions consisting of single cells; and a small number of budding cells in a population that has reached a stationary stage of growth. The cells were grown before irradiation up to stationary phase on a solid nutrient medium, and then, they were washed off with distilled water and resuspended to make a stock solution. The length of preirradiation growth was determined by cell budding cease.
Irradiation with ionizing radiations and ultraviolet light
Cells from the same suspension were exposed to 60 Co γ-rays (LET = 0.2 keV/μm, 20 Gy/min) and 239 Pu α-particles (25 Gy/min). The γ-ray dose rate was measured with a calibrated Siemens ionization chamber. The LET of α-particles reaching a cell monolayer was estimated to be of 120–130 keV/μm. Exactly, at about this LET value, the maximum of RBE-LET relationship was observed for most eukaryotic and some prokaryotic unicellular organisms. The α-particle dose rate was determined by measuring the intensity and energy of the particles with a semiconductor silicon surface barrier detector at a distance corresponding to the cells. Procedure for α-irradiation was similar to that described earlier., The small range of α-particles necessitated the use of a monolayer of the yeast cells for α-irradiation: 0.02 ml of cell suspension was placed on the surface of a nonnutrient agar and the water from this drop of suspension was evaporated. All irradiations were carried out at room temperature (20°C ± 2°C).
The cell suspension (2 ml, 106 cells/ml) was irradiated in an open quartz vessel with germicidal lamp that emitted predominantly UV light of wavelength 254 nm (far-UV radiation) at 1.5 W/m 2. The fluence rate was measured using a calibrated General Electric Germicidal Meter. To avoid photoreactivation, irradiation, dilution of suspensions, and other procedures were performed in red light, while postradiation incubation of cells occurred in dark conditions.
Survival and genetic instability assays
After the treatment, the samples were diluted to the appropriate cell concentration and a known number of cells were plated in such a manner that survival cells produced 50–300 colonies. Survival response was determined on the basis of the colony counts obtained at the end of 2–3 days of incubation at 30°C and the counts checked again after a further period to ensure that the final score had been reached.
To quantify the delayed formation of colonies by cells surviving after irradiation, the colony grown in Petri dish More Detailses was counted after 22 h and every 2–6 h later until the colonies ceased to appear. The last count was made after 4 days. As a test of genetic instability, the percentage of colonies formed later than in control was used. The correspondence of such test to genetic instability was confirmed earlier , by a number of factors – cells form colonies produced later than in control were characterized by increased radiosensitivity and thermosensitivity, increased content of morphologically altered colonies, increased content of nonviable cells inside late-formed colonies, and an increased content of respiratory and recombinant mutants. Each experiment was repeated 2–5 times. The results are presented as an average and its standard error. Points in all figures are the means from at least three independent experiments. Error bars in all the figures indicate the standard errors of the mean.
| Results|| |
[Figure 1] shows the dependence of cell survival on exposure dose (further for brevity simply “survival curves”) for haploid (open circles) and diploid (closed circles) yeast cells exposed to γ-rays [Figure 1]a, α-particles [Figure 1]b, and UV light [Figure 1]c. It is seen that after irradiation with sparsely ionizing radiation [Figure 1]a, haploid cells exhibit exponential survival curve while diploid cells showed sigmoid inactivation kinetics and were much more resistant in comparison with haploids (6.2 times at 10% survival). This difference is usually interpreted as being due to repair of DNA DSBs that requires two homologous DNA duplexes. The exponential shape of survival curve for haploid cells indicates a less efficiency of recovery from radiation-induced damage. The ability of haploid cells to recover DSB is now also known by virtue of discovery of repair by nonhomologous end joining, the effectiveness of which is much lower than the recovery through the recombination of homologous chromosomes.
|Figure 1: Survival curves of wild-type haploid (curves 1) and diploid (curves 2) Saccharomyces cerevisiae yeast cells exposed to 60Co γ-rays (panel A), 239Pu α-particles (panel B), and 254 nm ultraviolet light (panel C)|
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After exposure to α-particles [Figure 1]b, the shape of survival curves of haploid and diploid cells stayed without change in comparison with gamma irradiation and diploid cells remained more resistant than haploids although the degree of curvature of the initial part of diploid survival curve was slightly decreased. The difference in their radioresistance also decreased (2.1 times at 10% survival). This was due to the fact that high-LET radiations produce a greater proportion of irreversible damage, from which cells are unable to recover. In this paper, it was shown that the irreversible component was enhanced for the densely ionizing radiation in comparison to the low LET radiation while the probability of the recovery was identical for both the low and high LET radiations for several of yeast strains investigated. It was concluded that the recovery process itself is not damaged after densely ionizing radiation and the enhanced RBE of the high-LET radiation may be caused by the increased yield of the irreversible damage.
Irradiation with UV light [Figure 1]c exhibited somewhat different response – the shape of survival curves remained sigmoid regardless of cell ploidy and haploid yeast cells irradiated with UV light were only 1.7 times more sensitive to UV light than diploid one.
As noted above, genetic instability, determined by the delayed appearance of colonies by irradiated cells, is more pronounced for diploid cells, regardless of the shape of the survival curve and the ability of cells to recover. In order to confirm this conclusion, it was of interest to compare genetic instability induced by various kinds of radiation in haploid and diploid cells. [Figure 2] shows the delayed appearance of clones by haploid (curves 1) and diploid (curves 2) yeast surviving after exposure to γ-rays [Figure 2]a, α-particles [Figure 2]b, and UV light [Figure 2]c. It can be seen that as the dose of the influencing factor increases, the proportion of surviving diploid cells forming colony later than in control increases and reaches almost 100% only for diploid cells irrespective of the type of radiation. This indicates that as the dose increases, the proportion of cells containing ineffective sublesions, responsible for the delayed appearance of clones, significantly increases. It can be seen that the genetic instability of haploid yeast cells, also characterized by a sigmoid survival curve for UV irradiation, is much less pronounced than in diploid cells, and is about 30%. This confirms our earlier conclusion  that genetic instability is mainly determined by cell ploidy rather than by the shape of survival curves. It is also seen that the delayed clone appearance increased more sharply with dose of α-irradiation of particles than γ-rays that indicates a greater efficiency of manifestation of the detected effect after the action of densely ionizing radiation. The RBE of α particles, determined by the ratio of the isoeffective doses of γ- and α-radiations, was 4.2 ± 0.2 in the range of the measured parameter change from 20% to 60%, i.e., α-particles are 4.2 times more effective than the action of sparsely ionizing radiation, which corresponds to the RBE of α-particles, estimated by the criterion of cell survival in this paper and reported in other reports.,, The same increased efficiency of α-particles for cell inactivation and the delayed formation of clones by surviving cells may be associated with greater efficiency of the densely ionizing radiation to produce lethal radiation damage and the corresponding sublesions that persist in distant descendants of surviving cells, thereby destabilizing the genome, which can manifest themselves in the slowing down of their reproduction.,
|Figure 2: Delayed appearance of colonies produced by haploid (curves 1) and diploid (curves 2) Saccharomyces cerevisiae yeast cells surviving after irradiation with 60Co γ-rays (panel A), 239Pu α-particles (panel B), and 254 nm ultraviolet light (panel C)|
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In order to compare the effectiveness of ionizing radiation action on genetic instability with the influence of UV light on this effect, we presented the dependence of the delayed colony formation on cell survival for haploid and diploid yeast cells [Figure 3]. This figure contains data for the late appearance of colonies by cells surviving after irradiation with γ-rays, α-particles, and UV light. The obtained results show that the delay in the formation of colonies by irradiated cells in the dependence on their survival was practically independent of kind of radiation. These data indicate that equally effective lethal damage is accompanying by equal number of sublesions responsible for genetic instability. In other words, this effect is associated with the accumulation of sublesions ineffective for cell inactivation. We can note again that these sublesions are preserved in the remote descendants of surviving cells after irradiation and may manifest themselves in slowing down of their reproduction., This problem is important in connection with the use of synergy ideas  in the treatment of tumors in photoradiation and photochemotherapy.
|Figure 3: The relationship between cell survival and delayed appearance of colonies produced by haploid (curve 1) and diploid (curve 2) Saccharomyces cerevisiae yeast cells surviving after irradiation with 60Co γ-rays (circles), 239Pu α-particles (triangles), and 254 nm ultraviolet light (squares)|
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| Discussion|| |
New data concerning the delayed appearance of clones by cells surviving after irradiation with UV-light, sparsely and densely ionizing radiation are presented in this work. The results obtained for survival [Figure 1] and delayed appearance of clones [Figure 2] by isogenic haploid and diploid S. cerevisiae yeast cells surviving after exposure to various radiations exemplifying genetic instability are partly confirmed by previously published data,,, indicating this effect is more pronounced for diploid than for haploid yeast strains (100% vs. 20%). It was asserted in the cited papers that genetic instability is natural for diploid yeast cells with sigmoid shape of dose-response curves due to cell ability to recover from radiation damage, while this effect was negligible or even not observed for strains with exponential survival curves. It might be the time to shift this paradigm. The authors of these papers did not use radiosensitive mutants with exponential survival curves both for haploid and diploid yeast cells exposed to low- and high-LET radiations. However, it was shown recently  that genetic instability defined by the delayed appearance of clones was well expressed and reached 100% for diploid cells irrespectively of the shape of survival curve and cell ability to recover from radiation damage. This effect was poorly expressed (20%–30%) for all haploid strains characterized by an exponential survival curve. Consequently, the effect of the delayed appearance of clones by diploid yeast cells surviving after irradiated is not uniquely related to the shape of survival curve and cell ability to recover from radiation damage. In other words, the presence of a diploid-specific repair  is not the main determinant of the late appearance of colonies by irradiated cells. This point of view is supported by new data presented here on the genetic instability of yeast cells after UV irradiation. Obviously, despite the sigmoid form of survival curves of both haploid and diploid yeasts, the delayed appearance of clones is more pronounced for diploid but not for haploid cells (100% vs. 30%). Thus, the data presented here and some of our earlier data  show that the delayed appearance of clones produced by cells surviving after irradiation with ionizing radiations of different quality and UV light is mainly determined by cell ploidy rather than by the sigmoid form of survival curve and cell ability to recover radiation damage, as repeatedly postulated.,,
Another important new fact obtained here is that the RBE values of α-particles are approximately the same for both test effects studied with haploid (RBE = 2.0 ± 0.2) and diploid (RBE = 4.2 ± 0.3) cells. Identity of RBE values for cell survival and genetic instability may be associated with the greater efficiency of densely ionizing radiation to produce lethal radiation damage and accompanying sublesions responsible for the delayed appearance of clones by cells surviving after irradiation. Sublesions produced are preserved in the remote descendants of cells surviving after irradiation and thereby cause destabilization of genome, which is manifested in the delayed appearance of clones after irradiation. These RBE values for cell survival have been published by many authors ,,,, while the RBE magnitude for genetic instability are presented for the first time. The explanation of these results can be originated from the fact that high-LET radiations are known to produce clustering of ionizations and excitations in DNA, which produce more severe and complex damage than low-LET reference radiation. This also agrees with our earlier results  that densely ionizing radiation inhibits cell recovery due to greater fraction of irreversible damage, while the recovery constant did not depend on radiation quality.
The dependence of the delayed colony appearance by haploid and diploid yeast cells on their survival [Figure 3] was substantially the same after exposure to UV light, sparsely and densely ionizing radiation. On this basis, a nontrivial conclusion is proposed consisting in that the number of the produced radiation damage responsible for cell death may be directly proportional to the number of some sublesions accountable for genetic instability.
Many mechanisms have been proposed on how radiation exposure can give rise to genetic instability., It is beyond the scope of this paper to discuss in detail the status of molecular mechanisms underlying genetic instability described here although some results are worthy of mention. There is evidence that repair of DNA DSB is important for the induction of delayed survival and reproductive death. Eukaryotic cells possess two DNA DSB pathways: homologous recombination , and nonhomologous DNA end joining., It was supposed that the repair of radiation-induced DNA DSBs may initiate the genomic instability and carcinogenesis., It was noted that mitotic recombination can serve as one of the ways of genome instability initiation. The increased frequency of mitotic recombination may correspond to cells that received upon irradiation some sublesions that persisted in distant descendants of cells surviving after irradiation and responsible for the delayed appearance of clones. Since in our paper  the genetic instability (~100%) was demonstrated for diploid yeast strains defective in recombination (rad 51/rad 51, rad 52/rad 52), it may be reasonably assumed that homologous recombination cannot be considered as a reason for the delayed appearance of clones observed here. It may be concluded that mechanism of the genetic instability was mainly determined by cell ploidy, independent on cell ability to recover from radiation damage and could be conceivably related with some chromosomal damages, which are lethal for haploid but not for diploid cells. The nature of such kind of damages remains to be elucidated.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]