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Chromosomal aberrations and micronuclei induced in onion (Allium cepa) by gamma-radiation
Journal of Environmental Radioactivity 207 (2019) 1–6
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Chromosomal aberrations and micronuclei induced in onion (Allium cepa) by gamma-radiation
T
A. Bolsunovskya,∗, D. Dementyeva, E. Trofimovaa, E. Iniatkinaa, Yu Kladkob, M. Petrichenkovc a
Institute of Biophysics Siberian Branch of Russian Academy of Sciences, 50-50 Akademgorodok, Krasnoyarsk, 660036, Russia Institute of Forest Siberian Branch of Russian Academy of Sciences, 50-28 Akademgorodok, Krasnoyarsk, 660036, Russia c Budker Institute of Nuclear Physics of the Siberian Branch of the Russian Academy of Sciences, 11 Lavrentyev Ave., Novosibirsk, 630090, Russia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Allium cepa Chromosomal aberrations Micronuclei Gamma-radiation The dose-response curve Low doses Germinating seeds
The Allium-test is commonly used to assess genotoxicity of chemical and physical factors. In the present study, the roots of germinating onion (Allium cepa) were exposed to 0.02–13 Gy of γ-radiation. The dose dependencies of the frequency of chromosomal aberrations and micronuclei were nonlinear. An increase in the frequency of chromosomal aberrations in germinating seed root cells was first found under exposure to low doses of γ-radiation (0.05 and 0.1 Gy). Micronuclei inductions at low doses of radiation were not significantly different from the control. Our study suggests that germinating onion seed roots are a sensitive bioassay material for assessing the genotoxic effects of low-dose γ-radiation.
1. Introduction Nuclear weapons tests, long-term operation of nuclear power plants, and accidents that have occurred at them have released considerable amounts of artificial radioactivity into the environment. This has caused an increase in radiation exposure of organisms in some areas. The floodplain of the Yenisei River is contaminated by artificial radionuclides, including those associated with radioactive microparticles released by the Mining-and-Chemical Combine of Rosatom, which has been in operation for many years (Bolsunovsky, 2010; Bolsunovsky and Tcherkezian, 2001; Bolsunovsky et al., 2017a). Radioactive particles with high 137Cs activity (up to 30 MBq) (Bolsunovsky and Tcherkezian, 2001; Bolsunovsky et al., 2017a) are point sources of external γ-and β-radiation and are responsible for additional radiation exposure of aquatic and terrestrial organisms. In the Yenisei River ecosystem, organisms are exposed primarily to low doses of ionizing radiation (Bolsunovsky et al., 2005). In our previous studies, we modeled the effect of γ-radiation from radioactive particles in laboratory experiments with various plant and bacterial bioassays, which showed their high sensitivity to low-dose γradiation (Bolsunovsky et al., 2016a, 2017b). In some of our toxicological studies, we used the Allium-test (Bolsunovsky et al., 2016b), which is suggested as a standard for assessment of the chemical and radiation toxicity of environmental samples (Geras'kin et al., 2011; Hoshina and Marin-Morales, 2009; Kovalchuk et al., 1998; Leme and
∗
Marin-Morales, 2009). Our experiments with the Allium-test used to assess the toxicity of ionizing radiation revealed stimulation rather than inhibition of the growth of roots exposed to the experimental doses (Bolsunovsky et al., 2016b). In the standard Allium-test, both onion bulbs and seeds can be used (Kovalchuk et al., 1998; Tkalec et al., 2009; Takatsuji et al., 2010). It is convenient to use seeds, as they are biologically dormant and genetically and physiologically uniform test objects. Apical root meristems, as proliferating tissues, have high sensitivity to the genotoxic effects of physical and chemical factors. The chromosomal aberration test and the micronucleus test are known as efficient genetic bioassays. Genetic bioassays using onion seeds showed good results in assessing cyto- and genotoxicity of γ-radiation and fast neutrons (Amjad and Anjum, 2003; Zhang et al., 2003), high energy heavy ions (Takatsuji et al., 2010), and electromagnetic radiation (Tkalec et al., 2009). Those studies, however, used high doses of radiation, and, thus, no dose dependencies were found for the cytogenetic parameters of germinating seed roots exposed to low doses of γ-radiation. The purpose of this study was to assess the effects of γ-radiation, including low-dose radiation, on cytogenetic parameters of germinating seed roots in onion (Allium cepa).
Corresponding author. Institute of Biophysics, Siberian Branch of Russian Academy of Sciences, 50-50 Akademgorodok, Krasnoyarsk, 660036, Russia. E-mail addresses: [email protected], [email protected] (A. Bolsunovsky).
https://doi.org/10.1016/j.jenvrad.2019.05.014 Received 30 October 2018; Received in revised form 6 May 2019; Accepted 20 May 2019 0265-931X/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Chromosomal aberrations in cells of germinating onion seed roots as dependent on absorbed dose in experiments conducted in 2016 and 2017. Dose (Gy)
2016 Control 0.02 0.05 0.1 1 3 5 2017 Control 0.1 1 2.6 4.5 6.4 13
Total aberrations (%)
Individual aberrations of different types (%) Bridges Fragments Lagging chromosomes
Other
Multiple aberrations of different types (%) Total multiple Bridges Fragments aberrations (%)
Lagging chromosomes
4.6 ± 1.2 5.2 ± 2.0 7.7 ± 2.8 6.5 ± 1.6 22.3 ± 4.6 55.5 ± 6.1 74.7 ± 5.2
1.4 1.5 2.6 2.4 4.5 6.2 7.0
0.2 0.1 0.6 0.3 2.0
0.6 ± 0.4 0.9 ± 0.5 0.8 ± 0.6 0.7 ± 0.4 3.1 ± 1 14.7 ± 5.8 33.1 ± 5.1
1.9 ± 0.8 2.2 ± 1.1 3.3 ± 1.1 3.1 ± 1.2 6.4 ± 0.2 13.8 ± 5.3 16.7 ± 6.3
1.8 ± 1.1 2.4 ± 1.1 3 ± 1.9 2.6 ± 1 7.4 ± 2.1 21.5 ± 2.7 34.4 ± 5.5
1.3 ± 0.5 1.7 ± 1.1 1.9 ± 1.8 1.5 ± 0.8 10.9 ± 3.6 32.9 ± 5 52.1 ± 6
1.8 ± 1.8 2.5 ± 1.9 10.4 ± 4.3 33.3 ± 10.9 57.4 ± 15.9 75.7 ± 21.3 91.4 ± 16.2
1.0 ± 1.4 1.5 ± 1.4 3.5 ± 2.4 5.6 ± 3.6 6.1 ± 4.3 18.2 ± 16.1 16.0 ± 24.8
0.5 1.9 5.2 2.8
0.1 ± 0.4 0.1 ± 0.3 0.9 ± 1.0 8.7 ± 3.5 19.4 ± 8.9 28.3 ± 16.6 58.9 ± 27.6
1.2 ± 1.5 1.6 ± 1.5 4.2 ± 2.4 9.3 ± 4.0 12.1 ± 7.7 33.1 ± 19.1 52.6 ± 26.5
0.4 ± 0.7 0.7 ± 1.3 2.1 ± 1.5 12.2 ± 7.0 27.1 ± 14.9 28.2 ± 20.6 32.7 ± 33.7
0.3 ± 0.6 0.3 ± 0.6 4.9 ± 2.7 20.5 ± 8.4 38.9 ± 11.5 43.3 ± 20.1 56.0 ± 31.0
± ± ± ± ± ± ±
0.6 0.8 1.1 1.1 0.6 1.9 2.4
1.5 ± 0.8 1.7 ± 0.9 2.4 ± 1.6 2.1 ± 0.9 5.4 ± 1.3 11.6 ± 0.9 8.4 ± 0.4
0.9 ± 0.5 1.0 ± 0.7 1.5 ± 1.3 1.1 ± 0.8 8.6 ± 3.0 21.1 ± 4.6 23.6 ± 8.7
0.2 — 0.3 0.1 0.7 1.8 2.5
0.3 0.6 1.9 5.8 9.8 6.4 0.5
0.3 ± 0.6 0.3 ± 0.6 4.1 ± 2.5 13.1 ± 6.6 21.5 ± 9.2 20.1 ± 15.3 14.8 ± 18.0
— — — 0.2 0.8 2.8 1.2
± ± ± ± ± ± ±
0.6 1.3 1.4 5.8 8.0 9.9 2.1
2. Materials and methods
± 0.2 ± ± ± ± ±
± ± ± ±
aberrations served as an indicator of genotoxicity. Between 400 and 1200 cells were scored in each treatment for different exposure doses. Chromosomal aberrations (single and double bridges; fragments, stickiness), mitotic aberrations (vagrant, including lagging, chromosomes, multipolar mitosis, unequal disjunction of chromosomes), and multiple aberrations were identified in anaphase–telophase cells. The frequency of cells with chromosomal aberrations was determined as the ratio of the number of cells with aberrations to the total number of anaphase-telophase cells examined. The presence of micronuclei (the frequency of cells with micronuclei) was determined in interphase cells. Approximately 1000 cells per slide were evaluated and 5 slides per treatment (in total, 5000 cells per treatment). The frequency of cells with micronuclei was determined as the ratio of the number of cells with micronuclei to the total number of interphase cells examined.
2.1. Plant materials and irradiation procedure Seeds of onion (Allium cepa L. cv. Stuttgarter Riesen) were used in γradiation bioassay. The seeds were preliminarily germinated in polypropylene containers, on filter paper wetted with distilled water. Germinating seeds with 2-3-mm-long roots were taken for experiments. The germinating seeds were placed into transparent polypropylene containers, onto a bed of two layers of filter paper wetted with distilled water. Fifteen germinating seeds were used for each level of radiation and for the control. Experiments were conducted for 24 h at a temperature of 18–21 °C, in the dark. During 2016–17, seven experiments were conducted. A point source containing 137Cs (T1/2 = 30.17 yr) in a steel capsule, which emitted γ-quanta at the energy of 661.66 keV, was used as a source of external γ-radiation for germinating onion seeds at the G.I. Budker Institute of Nuclear Physics SB RAS (Novosibirsk). The radiation dose to germinating onion seeds was varied by placing the containers with the seeds at different distances from the radiation source. In experiments carried out in 2016, the dose rate of γ-radiation was varied within a range of 0.8–208 mGy h−1, and the adsorbed doses for germinating seeds were 0.02, 0.05, 0.1, 1, 3, and 5 Gy. In experiments of 2017, the dose rate of γ-radiation was varied within a range of 4–530 mGy h−1, and the adsorbed doses for germinating seeds were 0.1, 1, 2.6, 4.5, 6.4, and 13 Gy. The dose rate calculations were based on the activity of the 137Cs source; they were verified by direct measurements with a DKG-02U dosimeter (SPC “Doza”, Ltd, Russia). Unexposed germinating onion seeds were used as the control (the dose rate in the control was 0.002 mGy h−1).
2.3. Statistical analysis of data The experimental results were analyzed by the methods of variation statistics using the STATISTICA 7.0 software package. The statistical significance of differences was evaluated using Student's t-test. Figure and Table 1 give the means and standard deviations of the means. 3. Results In the experiments, the length of germinating seed roots varied nonsignificantly (between 8.5 ± 1.3 and 10.9 ± 1.9 mm) under exposure to γ-radiation doses from 0.02 to 13 Gy. The average root length at all exposure levels was 10.0 ± 1.7 mm and did not differ significantly from the average root length in the control (9.4 ± 1.8 mm). Even at the highest dose (13 Gy), the root length was 9.2 ± 1.4 mm and did not differ from the root length in the control either. Thus, all experiments showed that γ-radiation within a dose range between 0.02 and 13 Gy did not produce any statistically significant effect on the length of the germinating seed roots. Analysis of the frequency of cells of germinating onion roots with chromosomal aberrations showed a dose dependent increase in aberrations in all experiments. At the highest exposure dose, 13 Gy, aberrations were detected in the majority of anaphase and telophase cells, and the frequency of cells with chromosomal aberrations reached 91% (Fig. 1A, Table 1). In roots exposed to low doses (0.05 and 0.1 Gy), the frequencies of chromosomal aberrations were 7.7% and 5.3%, respectively, and they were significantly higher than in the control samples (Fig. 1A). The data of the dose–response curve in the portion between 0 and 6.4 Gy can be described by a linear equation: y = 12.12x + 6.15, R2 = 0.96. In the dose range between 5 and 13 Gy, the frequencies of cells with chromosomal aberrations did not differ significantly, i.e. the
2.2. Endpoints of germinating onion seeds The following endpoints of germinating onion roots were used as toxicity indicators: total chromosomal aberrations and different types of anaphase-telophase aberrations; micronuclei; and average length of germinating roots. For the cytogenetic study, roots were fixed in 96% ethyl alcohol: glacial acetic acid (3:1 v/v) immediately after exposure. After several days, the roots were stained with 1% acetic-orcein mixture (Medvedeva et al., 2014). Squashed slides were prepared from root tips, which were immediately analyzed by optical microscopy (Olympus CX31) at × 600 magnification and photographed. Cytogenetic abnormalities were studied using the method of Allium anaphase-telophase chromosome aberration assay and micronucleus test. The frequency of anaphase-telophase cells with chromosomal 2
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aberrations (data presented in the column “Other” of Table 1) and were less informative than the three major types of aberrations. In experiments carried out in 2017, in addition to analysis of chromosomal aberrations and the length of germinating seed roots, we conducted a micronucleus test of the apical meristem cells of germinating seed roots. The frequency of micronuclei in interphase cells of germinating seed roots exposed to a dose of γ-radiation of 1 Gy or higher was significantly different from the control (Fig. 1B). In the cells exposed to a low dose of 0.1 Gy, the frequency of micronuclei (1.2 ± 0.5%) was higher than in the control (0.6 ± 0.5%), but the difference was non-significant. That suggested lower sensitivity of the micronucleus test compared to the chromosomal assay for the cells of germinating onion seed roots exposed to low-dose radiation. The data in the dose-response curve for the micronuclei in the range between 0 and 4.5 Gy, can be described by the linear equation y = 3.27x+0.94, R2 = 0.99, as in the analysis of chromosomal aberrations. In the dose range between 2.6 and 13 Gy, the frequencies of cells with chromosomal aberrations did not differ, i.e. the dose–response curve exhibited a plateau. All data in the dose-response curve of the frequency of micronuclei can be approximated by the second-degree polynomial equation: y = – 0.24x2 + 3.50x + 1.27, R2 = 0.85 (Fig. 1B). The dose-response curves of the frequency of the micronuclei and the frequency of chromosomal aberrations had similar shapes, with an initial linear segment followed by a plateau, when there was no dose dependence. The plateau for the frequency of chromosomal aberrations was in the dose range between 5 and 13 Gy, while the plateau for the frequency of micronuclei was broader, extending from 2.6 to 13 Gy. At the highest dose, 13 Gy, the average frequency of micronuclei decreased, but it was not significantly different from the frequencies at lower doses (Fig. 1B). We performed correlation analysis to determine the relationship between the frequency of cells with micronuclei and the levels of different types of chromosomal aberrations (using the data of 2017 in Table 1). The frequency of micronuclei was significantly correlated to the levels of individual chromosomal aberrations such as fragments (r2 = 0.91 p < 0.01) and lagging chromosomes (r2 = 0.95 p < 0.01). When individual chromosomal aberrations were combined with the same types of abnormalities in the multiple aberrations, the frequency of micronuclei was significantly correlated only to the total level of fragments (r2 = 0.77 p < 0.05) but was not correlated to the total lagging chromosomes. Thus, the study of the chromosomal aberrations suggests that fragments are likely the main source of formation of micronuclei in cells of germinating seed roots.
Fig. 1. Absorbed dose (Gy) dependence of the frequencies of chromosomal aberrations (A) and micronuclei (B) in the cells of germinating onion seed roots (%). Approximation of the data with linear (1) and second-degree polynomial (2) models. Line (3) is the control. * – statistically significant difference from the control level (p < 0.05).
dose–response curve exhibited a plateau. All data in the dose-response curve of the frequency of chromosomal aberrations can be approximated by the second-degree polynomial equation: y = – 0.74x2 + 16.30x + 4.29, R2 = 0.98 (Fig. 1A). Three types of aberrations – bridges, fragments, and lagging chromosomes – made the major contribution to the total chromosomal aberrations (Table 1). Among these individual aberrations, the percentage of lagging chromosomes was somewhat higher (more than 20%), but at an exposure dose of 6.4 Gy or more, the percentages of lagging chromosomes and the bridges were virtually the same. The fragments constituted no more than 11%. As the exposure dose was raised, the frequencies of almost all types of chromosomal aberrations increased. However, at a dose of 5 Gy or higher, the percentage of fragments declined, reaching its minimum at the highest dose – 13 Gy (Table 1). In addition to these three types of individual aberrations, we detected multiple aberrations: two or three aberrations of the types mentioned above in a cell. At the highest exposure doses (6.4 and 13 Gy), the frequency of multiple aberrations was higher than the frequency of individual aberrations, reaching 28–59% (Table 1). An exposure dose of 2.6 Gy can be considered as the threshold of the occurrence of multiple aberrations: at this dose, multiple aberrations increased by an order of magnitude compared to aberrations at the dose of 1 Gy (from 0.9 to 9%). As the exposure dose was raised, an increase, similar to that observed in the main types of individual aberrations, was detected in the three types of multiple chromosomal aberrations. However, at doses of 3–4.5 Gy or higher, the percentage of fragments remained unchanged, in contrast to the other two types of aberrations (Table 1). In addition to the three major types of aberrations, we also detected chromosomal stickiness, multipolar and asymmetric mitoses, and ring chromosomes, but they constituted no more than 3% of the total
4. Discussion The standard Allium-test (with onion bulbs) has been traditionally used to assess chemical toxicity (Leme and Marin-Morales, 2009; Hoshina and Marin-Morales, 2009: Seth et al., 2008) while the toxicity of ionizing radiation has been studied far less extensively. A number of researchers (Cortés et al., 1990; Kumar et al., 2011; Perez-Talavera et., 2003; Sinovets et al., 2009; Vaijapurkar et al., 2001) reported that the standard Allium-test with bulbs can be effectively used to assess toxicity of ionizing radiation to such endpoints as root length, chromosomal aberrations, micronuclei, and mitotic index only at relatively high exposure doses (above 1.5–2 Gy). At low exposure doses (≤0.1 Gy), no inhibition of onion endpoints was observed, but two studies (Sinovets et al., 2009; Bolsunovsky et al., 2016b) detected stimulation of endpoints. As already noted, the Allium-test can be conducted with both bulbs and seeds. Moreover, seeds are more convenient test objects, as they are biologically dormant and genetically and physiologically uniform. Kovalchuk et al. (1998) reported a study in which onion seeds were used to assess genotoxicity of soil samples from the zone affected by the Chernobyl NPP. The authors found correlation between the total chromosomal aberrations in root cells of seedlings and content of 137Cs, 3
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frequency of micronuclei in the cells of germinating onion seed roots began in the range of 2.6–4.5 Gy, and that was consistent with the data reported for the micronuclei of the irradiated onion bulbs (George et al., 2014). The plateau for the frequency of the chromosomal aberrations in our experiments began at 5–6.4 Gy, i.e. close to the plateau for micronuclei (Fig. 1). At high exposure doses, however, we did not obtain another significant linear change in the frequency of micronuclei, which had been observed in the third phase in the study by George et al. (2014). Thus, we obtained a bi-phasic rather than a tri-phasic dose–response curve. In experiments with γ-irradiation of Allium sativum bulbs, George et al. (2014) obtained a quadri-phasic rather than a triphasic dose–response curve: between the dose-independent plateau and the second linear micronuclei frequency increase, there was a phase of a decrease in the frequency of micronuclei in the cells of Allium sativum roots. Our findings and the data reported in the study cited above suggested that different Allium species and different stages of development (bulbs and germinating seeds) of the same species (Allium cepa) showed dissimilar responses to γ-irradiation. In a study by Takatsuji et al. (2010), the authors exposed germinating onion seeds to three types of high energy accelerated heavy ions and observed micronuclei in the root tip cells. An increase in micronuclei induction was first recorded at 0.05 Gy, reaching its maximum at 1.0–1.5 Gy. In the dose range between 1 and 2 Gy, micronuclei induction either remained unchanged or decreased depending on the type of high energy heavy ions, i.e. bi- and tri-phasic dose–response curves were obtained. In the control, however, micronuclei induction was 0.4% (similar to our results), and at the highest exposure doses it reached 20–42%, depending on the type of high energy accelerated heavy ions. In that study, micronuclei induction was described with a dose response curve of an upward convex bell shape. The decrease in micronuclei frequency at high doses was caused by the lethal damage to cells done by heavy ions. The data discussed above show that the nonlinear dose dependence of cytogenetic abnormalities is usually characterized by a tri-phasic dose–response curve. However, some of the studies report bi-phasic dose–response curves (the present work and some of the curves in research by Takatsuji et al. (2010)) and quadri-phasic ones (George et al., 2014). Authors cited above (Geras'kin, 1995; Geras'kin et al., 2007; George et al., 2014) attribute the nonlinear dose dependence of cytogenetic abnormalities to differences in the status of DNA repair processes, with DNA repair activated by a certain threshold dose or level of DNA damage. At low doses, the effects of exposure to radiation are minor compared to spontaneous ones. Hence, the increase in micronuclei induction or chromosomal aberrations at the lower doses representing the first linear phase of the dose curve was associated with misrepair events. The following phase is plateau, when in spite of a considerable dose increase, no change is observed in the frequency of micronuclei or chromosomal aberrations. In that phase, as the dose is increased, repair mechanisms are activated in response to the radiation-induced DNA damage. As suggested by George et al. (2014), the DNA repair mechanisms may be activated by a threshold dose or level of DNA damage. In our experiments, 2.6 Gy may be the threshold dose activating DNA repair mechanisms, and this dose is similar to the threshold dose (2 Gy) reported by George et al. (2014). At the end of the plateau phase, as the dose is increased and the repair mechanisms become more effective, genetic damage may even be reduced. Usually, however, at a higher dose, the effectiveness of the repair systems decreases and, finally, they are suppressed, and the third phase of linear dose-dependent increase in aberrations is observed (George et al., 2014). As noted above, there are no available literature data on inhibition of the endpoints of onion (bulbs and seeds) by low doses of γ-radiation (≤0.1 Gy). The only study reporting irradiation of onion (Allium cepa) bulbs with γ-radiation (George et al., 2014) demonstrated that a significant increase in micronuclei induction relative to the control was only observed at the lowest dose, 0.7 Gy. That study on micronuclei
but they presented no data on absorbed doses of the seedling roots. The study also reported that the major aberrations contributing to the highest total percentage of chromosomal aberrations were bridges and fragments, which were the prevailing types of chromosomal aberrations in our experiments (Table 1). Tkalec et al. (2009) investigated the effect of electromagnetic fields (EMFs) on the cytogenetic and growth parameters of germinating onion seed roots. The authors showed that a more than tenfold increase in EMFs did not affect the length of the roots (like in our experiments), suggesting low sensitivity of physiological parameters of the roots to EMFs. However, the total chromosomal aberrations increased from 1 to 2% in the control to 4–7% in the treatment. EMFs induced such aberrations as lagging chromosomes, vagrants, disturbed anaphase, and chromosome stickiness. At the same time, the authors of that paper did not mention such aberrations as bridges and fragments. In our experiments, we irradiated germinating Allium cepa seed roots and obtained γ-radiation dose dependencies for such endpoints as the frequency of chromosomal aberrations and micronuclei. We have not found any data of other researchers on dose dependencies for the frequency of chromosomal aberrations after exposure of germinating onion seed roots to γ-radiation. There are, however, literature data on dose dependencies for the frequency of micronuclei after exposure of germinating onion seed roots to γ-radiation (Zhang et al., 2002, 2003; George et al., 2014) and high energy heavy ions (Takatsuji et al., 2010). The available data on radiation exposure of dry and wet seeds cannot be compared with the data on exposure of seedlings, as they exhibit different responses to radiation exposure. Rice (Oryza sativa, japonica) seeds were exposed to carbon ions at 20 Gy (Jin-Ming Shi et al., 2010), and that dose was lethal to the wet seeds and seedlings of rice while the dry seeds remained viable after the 20 Gy exposure. At a dose of 20 Gy, the frequency of chromosomal aberrations in root meristems of rice induced by carbon ion radiation was the highest for seedlings (22.5%) while for dry seeds and wet seeds it was considerably lower: 5.5 and 13%, respectively. The chromosomal aberrations in cells of rice seeds and seedlings included bridges, fragments, laggards, stickiness, and disturbed spindle. These types of chromosomal aberrations observed in cells of rice seedlings exposed to carbon ions are the same as the types of chromosomal aberrations in our experiments with germinating onion seed roots exposed to γ-radiation (Table 1). The difference in radiosensitivity between the dry and wet seeds may be caused by the difference in the water content. Roots of germinating seeds, as proliferating tissue meristems, have the highest sensitivity to the genotoxic effects of radiation factors. The data reported in a number of studies suggest the nonlinear dose dependence of the frequency of genetic disorders under exposure to low and medium doses of radiation. The authors of those studies (Geras'kin, 1995; Geras'kin et al., 2007; George et al., 2014) noted that the doseresponse curves showed not only a linear increase in genetic disorders but also a dose-independent part (a plateau). Such tri-phasic dose–response curves were obtained for various biological objects (Chinese hamster fibroblast cells and Vicia faba root cells, human lymphocytes, barley and onion root cells), suggesting that this behavior of the doseresponse curve could not be attributed to random experimental fluctuations but was rather caused by the similar responses of genetic structures to the dose increase. The dose-response curves obtained in the present study (Fig. 1) support the data cited above, which suggest nonlinear dose dependence of various genetic disorders in the region of low and medium exposure doses. The most noteworthy study was reported by George et al. (2014). The authors obtained nonlinear dependence of the frequency of micronuclei after irradiation of onion (Allium cepa) bulbs with γ-radiation. The linear increase in the micronuclei frequency in a range of doses between 0.7 Gy and 2 Gy was followed by a dose-independent plateau between 2 and 4 Gy, and then, at doses from 4 to 6 Gy, micronuclei induction increased again. In that study, the dose-independent response (plateau) began at 2 Gy. In our experiments, the plateau for the 4
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Acknowledgements
induction suggested that Allium cepa showed the lowest radiosensitivity among the 4 plant species studied. Similarly, in our experiments, the frequency of micronuclei in cells of germinating seed roots was significantly different from the control only at a dose of γ-radiation of 1 Gy or higher (Fig. 1B). However, in our experiments (Fig. 1A), the frequency of chromosomal aberrations showed the highest radiosensitivity to γ-radiation among the endpoints studied: at a low dose of 0.05 Gy or higher, it was different from the control. A similar conclusion, about the high radiosensitivity of the frequency of chromosomal aberrations to low-dose γ-radiation was made in a study by Geras'kin et al. (2007) for germinating spring barley (Hordeum vulgare) seeds. In that study, a triphasic dose–response curve was obtained for the frequency of chromosomal aberrations, and a significant increase in the frequency of chromosomal aberrations was observed at a dose of 0.05 Gy, like in the present study, for germinating onion seed roots (Fig. 1A). Chromosomal aberrations mentioned in the study by Geras'kin et al. (2007) were bridges, fragments, and lagging chromosomes. Lagging chromosomes made the largest contribution to the total chromosomal aberrations, and this is in good agreement with the results obtained in the present study (Table 1). That suggested high radiosensitivity of chromosomal assay compared to the micronucleus test for the cells of germinating onion seed roots exposed to low-dose radiation. The correlation analysis described above revealed significant positive correlation of the frequency of micronuclei to the levels of such chromosomal aberrations as fragments and, partly, lagging chromosomes in cells of germinating seed roots. Thus, the study of the chromosomal aberrations suggests that fragments are likely the main source of formation of micronuclei in cells of germinating seed roots. The review by Fenech et al. (2011) reported a study of the molecular mechanisms of micronucleus formation in human cells, which demonstrated that micronuclei can originate during anaphase from lagging acentric chromosomes or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Thus, in the interphase, the frequency of micronuclei is mainly determined by the frequency of chromosomal aberrations generated during anaphase. However, for plant cells, mechanisms of micronucleus formation remain insufficiently studied.
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Evidence for an adaptive response to radiation damage in plant cells conditioned with X-rays or incorporated tritium. Int. J. Radiat. Biol. 57 (3), 537–541. Fenech, M., Kirsch-Volders, M., Natarajan, A.T., Surralles, J., Crott, J.W., Parry, J., Norppa, H., Eastmond, D.A., Tucker, J.D., Thomas, P., 2011. Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26 (1), 125–132. https://doi.org/10.1093/mutage/ geq052. George, J.T., Patel, B.B., Rane, V.A., Venu-Babu, P., Eapen, S., Singhal, R.K., Pius, J., Narula, B., Shankhadarwar, S., 2014. Non-linear dose response of a few plant taxa to acute gamma radiation. Cytologia 79 (1), 103–109. Geras’kin, S.A., 1995. Concept of biological effect of low dose radiation on cells. Radiat. Biol. Radioecol. (Radiatsionnaya Biol. Radioekologiya) 35 (5), 563–580 (In Russian). Geras’kin, S.A., Oudalova, A.A., Kim, Jin Kyu, Dikarev, V.G., Dikareva, N.S., 2007. Cytogenetic effect of low dose γ-radiation in Hordeum vulgare seedlings: non-linear dose–effect relationship. Radiat. Environ. Biophys. 46, 31–41. Geras’kin, S., Oudalova, A., Michalik, B., Dikareva, N., Dikarev, V., 2011. Geno-toxicity assay of sediment and water samples from the Upper Silesia post-mining areas, Poland by means of Allium-test. Chemosphere 83, 1133–1146. Hoshina, M.M., Marin-Morales, M.A., 2009. Micronucleus and chromosome aberrations induced in onion (Allium cepa) by a petroleum refinery effluent and by river water that receives this effluent. Ecotoxicol. Environ. Saf. 72, 2090–2095. Jin-Ming Shi, Guo, Jian-Guo, Wen-Jian, L.I., Zhang, Meng, Huang, Lei, Sun, Ye-Qing, 2010. Cytogenetic effects of low doses of energetic carbon ions on rice after exposures of dry seeds, wet seeds and seedlings. J. Radiat. Res. 51, 235–242. Kovalchuk, O., Kovalchuk, I., Arkhipov, A., Telyuk, P., Hohn, B., Kovalchuk, L., 1998. The Allium cepa chromosome aberration test reliably measures genotoxicity of soils of inhabited areas in the Ukraine contaminated by the Chernobyl accident. Mutat. Res. 415, 47–57. Kumar, D.S., Chakrabarty, D., Arvind Kumar, Verma, Biresh Kumar, Banerji, 2011. Gamma ray induced chromosomal aberrations and enzyme related defense mechanism in Allium cepa L. Caryologia 64 (4), 388–397. Leme, D.M., Marin-Morales, M.A., 2009. Allium cepa test in environmental monitoring: a review on its application. Mutat. Res. 682 (1), 71–81. Medvedeva, M.Yu, Bolsunovsky, A.Ya, Zotina, T.A., 2014. Cytogenetic abnormalities in aquatic plant Elodea canadensis in anthropogenic contamination zone of Yenisei River. Contemp. Probl. Ecol. 7, 422–432. Perez-Talavera, S., Carballo, J.A., De La Torre, C., 2003. Unimpeded onset of proliferation and conserved processing of DNA damage in two Allium species after their challenge by ionizing radiation. Plant Biosyst. 137 (1), 11–20. Seth, C.S., Misra, V., Chauhan, L.K.S., Singh, R.R., 2008. Genotoxicity of cadmium on root meristem cells of Allium cepa: cytogenetic and Comet assay approach. Ecotoxicol. Environ. Saf. 71, 711–716. Sinovets, S.Yu, Pyatkova, S.V., Kozmin, G.V., 2009. Experimental validation of the use of the Allium-test in radioecological monitoring. News of higher educational institutions. Nucl. Energy 1, 32–38 (in Russian). Tkalec, M., Malari, K., Pavlica, M., Pevalek-Kozlina, B., Vidakovic-Cifrek, Z., 2009. Effects of radiofrequency electromagnetic fields on seed germination and root meristematic cells of Allium cepa L. Mutat. Res. 672, 76–81. Takatsuji, Toshihiro, Takayanagi, Hiroki, Morishita, Kana, Nojima, Kumie, Furusawa, Yoshiya, Nakazawa, Yuka, Matsuse, Michiko, Akamatsu, Sakura, Hirano, Natsuko, Hirashima, Natsuko, Hotokezaka, Saori, Ijichi, Toyomi, Kakimoto, Chika, Kanemaru, Tomomi, Koshitake, Mayumi, Moriuchi, Akiko, Yamamoto, Kensuke, Yoshikawa,
5. Conclusion The main effects of γ-radiation observed in the root cells of germinating onion (Allium cepa) seeds include an increase in the inductions of chromosomal aberrations and micronuclei. The dose dependencies of the frequency of chromosomal aberrations and micronuclei are nonlinear: the initial linear part is followed by a plateau, where no dose dependency is observed. The dose-independent plateau for the frequency of micronuclei begins at 2.6 Gy, and the plateau for the frequency of chromosomal aberrations begins at 5 Gy. An increase in the frequency of the chromosomal aberrations was first observed after exposure to γ-radiation in the low-dose range, suggesting high radiosensitivity of chromosomal assay compared to the micronucleus test for the cells of germinating onion seed roots exposed to low-dose radiation. In the high-dose region of radiation (up to 13 Gy), the length of the roots after exposure did not significantly differ from the length of the germinating seed roots in the control. Correlation analysis revealed significant positive correlation of the micronuclei frequency to the frequencies of such chromosomal aberrations as fragments and, partly, lagging chromosomes. These findings clearly demonstrated the genetic effects of γ-radiation, low doses in particular, on germinating onion seed roots. Therefore, cytogenetic endpoints of germinating onion seed roots can be used to assess biological effects of low-dose γ-radiation. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. 5
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root-tip cells of onion seedlings after irradiation as dry seeds. J. Radiat. Res. 43 (4), 397–403. Zhang, Wenyi, Fujikawa, Kazuo, Endo, Satoru, Ishikawa, Masayori, Ohtaki, Megu, Ikeda, Hideo, Hoshi, Masaharu, 2003. Energy-dependent RBE of neutrons to induce micronuclei in root-tip cells of Allium cepa onion irradiated as dry dormant seeds and seedlings. J. Radiat. Res. 44, 171–177.
Isao, 2010. Induction of micronuclei in germinating onion seed root tip cells irradiated with high energy heavy ions. J. Radiat. Res. 51, 315–323. Vaijapurkar, S.G., Agarwal, D., Chaudhuri, S.K., Senwar, K.R., Bhatnagar, P.K., 2001. Gamma-irradiated onions as a biological indicator of radiation dose. Radiat. Meas. 33 (5), 833–836. Zhang, Wenyi, Endo, Satoru, Ishikawa, Masayori, Ikeda, Hideo, Hoshi, Masaharu, 2002. Relative biological effectiveness of fission neutrons for producing micronuclei in the
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Chromosomal aberrations and micronuclei induced in onion (Allium cepa) by gamma-radiation
T
A. Bolsunovskya,∗, D. Dementyeva, E. Trofimovaa, E. Iniatkinaa, Yu Kladkob, M. Petrichenkovc a
Institute of Biophysics Siberian Branch of Russian Academy of Sciences, 50-50 Akademgorodok, Krasnoyarsk, 660036, Russia Institute of Forest Siberian Branch of Russian Academy of Sciences, 50-28 Akademgorodok, Krasnoyarsk, 660036, Russia c Budker Institute of Nuclear Physics of the Siberian Branch of the Russian Academy of Sciences, 11 Lavrentyev Ave., Novosibirsk, 630090, Russia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Allium cepa Chromosomal aberrations Micronuclei Gamma-radiation The dose-response curve Low doses Germinating seeds
The Allium-test is commonly used to assess genotoxicity of chemical and physical factors. In the present study, the roots of germinating onion (Allium cepa) were exposed to 0.02–13 Gy of γ-radiation. The dose dependencies of the frequency of chromosomal aberrations and micronuclei were nonlinear. An increase in the frequency of chromosomal aberrations in germinating seed root cells was first found under exposure to low doses of γ-radiation (0.05 and 0.1 Gy). Micronuclei inductions at low doses of radiation were not significantly different from the control. Our study suggests that germinating onion seed roots are a sensitive bioassay material for assessing the genotoxic effects of low-dose γ-radiation.
1. Introduction Nuclear weapons tests, long-term operation of nuclear power plants, and accidents that have occurred at them have released considerable amounts of artificial radioactivity into the environment. This has caused an increase in radiation exposure of organisms in some areas. The floodplain of the Yenisei River is contaminated by artificial radionuclides, including those associated with radioactive microparticles released by the Mining-and-Chemical Combine of Rosatom, which has been in operation for many years (Bolsunovsky, 2010; Bolsunovsky and Tcherkezian, 2001; Bolsunovsky et al., 2017a). Radioactive particles with high 137Cs activity (up to 30 MBq) (Bolsunovsky and Tcherkezian, 2001; Bolsunovsky et al., 2017a) are point sources of external γ-and β-radiation and are responsible for additional radiation exposure of aquatic and terrestrial organisms. In the Yenisei River ecosystem, organisms are exposed primarily to low doses of ionizing radiation (Bolsunovsky et al., 2005). In our previous studies, we modeled the effect of γ-radiation from radioactive particles in laboratory experiments with various plant and bacterial bioassays, which showed their high sensitivity to low-dose γradiation (Bolsunovsky et al., 2016a, 2017b). In some of our toxicological studies, we used the Allium-test (Bolsunovsky et al., 2016b), which is suggested as a standard for assessment of the chemical and radiation toxicity of environmental samples (Geras'kin et al., 2011; Hoshina and Marin-Morales, 2009; Kovalchuk et al., 1998; Leme and
∗
Marin-Morales, 2009). Our experiments with the Allium-test used to assess the toxicity of ionizing radiation revealed stimulation rather than inhibition of the growth of roots exposed to the experimental doses (Bolsunovsky et al., 2016b). In the standard Allium-test, both onion bulbs and seeds can be used (Kovalchuk et al., 1998; Tkalec et al., 2009; Takatsuji et al., 2010). It is convenient to use seeds, as they are biologically dormant and genetically and physiologically uniform test objects. Apical root meristems, as proliferating tissues, have high sensitivity to the genotoxic effects of physical and chemical factors. The chromosomal aberration test and the micronucleus test are known as efficient genetic bioassays. Genetic bioassays using onion seeds showed good results in assessing cyto- and genotoxicity of γ-radiation and fast neutrons (Amjad and Anjum, 2003; Zhang et al., 2003), high energy heavy ions (Takatsuji et al., 2010), and electromagnetic radiation (Tkalec et al., 2009). Those studies, however, used high doses of radiation, and, thus, no dose dependencies were found for the cytogenetic parameters of germinating seed roots exposed to low doses of γ-radiation. The purpose of this study was to assess the effects of γ-radiation, including low-dose radiation, on cytogenetic parameters of germinating seed roots in onion (Allium cepa).
Corresponding author. Institute of Biophysics, Siberian Branch of Russian Academy of Sciences, 50-50 Akademgorodok, Krasnoyarsk, 660036, Russia. E-mail addresses: [email protected], [email protected] (A. Bolsunovsky).
https://doi.org/10.1016/j.jenvrad.2019.05.014 Received 30 October 2018; Received in revised form 6 May 2019; Accepted 20 May 2019 0265-931X/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Chromosomal aberrations in cells of germinating onion seed roots as dependent on absorbed dose in experiments conducted in 2016 and 2017. Dose (Gy)
2016 Control 0.02 0.05 0.1 1 3 5 2017 Control 0.1 1 2.6 4.5 6.4 13
Total aberrations (%)
Individual aberrations of different types (%) Bridges Fragments Lagging chromosomes
Other
Multiple aberrations of different types (%) Total multiple Bridges Fragments aberrations (%)
Lagging chromosomes
4.6 ± 1.2 5.2 ± 2.0 7.7 ± 2.8 6.5 ± 1.6 22.3 ± 4.6 55.5 ± 6.1 74.7 ± 5.2
1.4 1.5 2.6 2.4 4.5 6.2 7.0
0.2 0.1 0.6 0.3 2.0
0.6 ± 0.4 0.9 ± 0.5 0.8 ± 0.6 0.7 ± 0.4 3.1 ± 1 14.7 ± 5.8 33.1 ± 5.1
1.9 ± 0.8 2.2 ± 1.1 3.3 ± 1.1 3.1 ± 1.2 6.4 ± 0.2 13.8 ± 5.3 16.7 ± 6.3
1.8 ± 1.1 2.4 ± 1.1 3 ± 1.9 2.6 ± 1 7.4 ± 2.1 21.5 ± 2.7 34.4 ± 5.5
1.3 ± 0.5 1.7 ± 1.1 1.9 ± 1.8 1.5 ± 0.8 10.9 ± 3.6 32.9 ± 5 52.1 ± 6
1.8 ± 1.8 2.5 ± 1.9 10.4 ± 4.3 33.3 ± 10.9 57.4 ± 15.9 75.7 ± 21.3 91.4 ± 16.2
1.0 ± 1.4 1.5 ± 1.4 3.5 ± 2.4 5.6 ± 3.6 6.1 ± 4.3 18.2 ± 16.1 16.0 ± 24.8
0.5 1.9 5.2 2.8
0.1 ± 0.4 0.1 ± 0.3 0.9 ± 1.0 8.7 ± 3.5 19.4 ± 8.9 28.3 ± 16.6 58.9 ± 27.6
1.2 ± 1.5 1.6 ± 1.5 4.2 ± 2.4 9.3 ± 4.0 12.1 ± 7.7 33.1 ± 19.1 52.6 ± 26.5
0.4 ± 0.7 0.7 ± 1.3 2.1 ± 1.5 12.2 ± 7.0 27.1 ± 14.9 28.2 ± 20.6 32.7 ± 33.7
0.3 ± 0.6 0.3 ± 0.6 4.9 ± 2.7 20.5 ± 8.4 38.9 ± 11.5 43.3 ± 20.1 56.0 ± 31.0
± ± ± ± ± ± ±
0.6 0.8 1.1 1.1 0.6 1.9 2.4
1.5 ± 0.8 1.7 ± 0.9 2.4 ± 1.6 2.1 ± 0.9 5.4 ± 1.3 11.6 ± 0.9 8.4 ± 0.4
0.9 ± 0.5 1.0 ± 0.7 1.5 ± 1.3 1.1 ± 0.8 8.6 ± 3.0 21.1 ± 4.6 23.6 ± 8.7
0.2 — 0.3 0.1 0.7 1.8 2.5
0.3 0.6 1.9 5.8 9.8 6.4 0.5
0.3 ± 0.6 0.3 ± 0.6 4.1 ± 2.5 13.1 ± 6.6 21.5 ± 9.2 20.1 ± 15.3 14.8 ± 18.0
— — — 0.2 0.8 2.8 1.2
± ± ± ± ± ± ±
0.6 1.3 1.4 5.8 8.0 9.9 2.1
2. Materials and methods
± 0.2 ± ± ± ± ±
± ± ± ±
aberrations served as an indicator of genotoxicity. Between 400 and 1200 cells were scored in each treatment for different exposure doses. Chromosomal aberrations (single and double bridges; fragments, stickiness), mitotic aberrations (vagrant, including lagging, chromosomes, multipolar mitosis, unequal disjunction of chromosomes), and multiple aberrations were identified in anaphase–telophase cells. The frequency of cells with chromosomal aberrations was determined as the ratio of the number of cells with aberrations to the total number of anaphase-telophase cells examined. The presence of micronuclei (the frequency of cells with micronuclei) was determined in interphase cells. Approximately 1000 cells per slide were evaluated and 5 slides per treatment (in total, 5000 cells per treatment). The frequency of cells with micronuclei was determined as the ratio of the number of cells with micronuclei to the total number of interphase cells examined.
2.1. Plant materials and irradiation procedure Seeds of onion (Allium cepa L. cv. Stuttgarter Riesen) were used in γradiation bioassay. The seeds were preliminarily germinated in polypropylene containers, on filter paper wetted with distilled water. Germinating seeds with 2-3-mm-long roots were taken for experiments. The germinating seeds were placed into transparent polypropylene containers, onto a bed of two layers of filter paper wetted with distilled water. Fifteen germinating seeds were used for each level of radiation and for the control. Experiments were conducted for 24 h at a temperature of 18–21 °C, in the dark. During 2016–17, seven experiments were conducted. A point source containing 137Cs (T1/2 = 30.17 yr) in a steel capsule, which emitted γ-quanta at the energy of 661.66 keV, was used as a source of external γ-radiation for germinating onion seeds at the G.I. Budker Institute of Nuclear Physics SB RAS (Novosibirsk). The radiation dose to germinating onion seeds was varied by placing the containers with the seeds at different distances from the radiation source. In experiments carried out in 2016, the dose rate of γ-radiation was varied within a range of 0.8–208 mGy h−1, and the adsorbed doses for germinating seeds were 0.02, 0.05, 0.1, 1, 3, and 5 Gy. In experiments of 2017, the dose rate of γ-radiation was varied within a range of 4–530 mGy h−1, and the adsorbed doses for germinating seeds were 0.1, 1, 2.6, 4.5, 6.4, and 13 Gy. The dose rate calculations were based on the activity of the 137Cs source; they were verified by direct measurements with a DKG-02U dosimeter (SPC “Doza”, Ltd, Russia). Unexposed germinating onion seeds were used as the control (the dose rate in the control was 0.002 mGy h−1).
2.3. Statistical analysis of data The experimental results were analyzed by the methods of variation statistics using the STATISTICA 7.0 software package. The statistical significance of differences was evaluated using Student's t-test. Figure and Table 1 give the means and standard deviations of the means. 3. Results In the experiments, the length of germinating seed roots varied nonsignificantly (between 8.5 ± 1.3 and 10.9 ± 1.9 mm) under exposure to γ-radiation doses from 0.02 to 13 Gy. The average root length at all exposure levels was 10.0 ± 1.7 mm and did not differ significantly from the average root length in the control (9.4 ± 1.8 mm). Even at the highest dose (13 Gy), the root length was 9.2 ± 1.4 mm and did not differ from the root length in the control either. Thus, all experiments showed that γ-radiation within a dose range between 0.02 and 13 Gy did not produce any statistically significant effect on the length of the germinating seed roots. Analysis of the frequency of cells of germinating onion roots with chromosomal aberrations showed a dose dependent increase in aberrations in all experiments. At the highest exposure dose, 13 Gy, aberrations were detected in the majority of anaphase and telophase cells, and the frequency of cells with chromosomal aberrations reached 91% (Fig. 1A, Table 1). In roots exposed to low doses (0.05 and 0.1 Gy), the frequencies of chromosomal aberrations were 7.7% and 5.3%, respectively, and they were significantly higher than in the control samples (Fig. 1A). The data of the dose–response curve in the portion between 0 and 6.4 Gy can be described by a linear equation: y = 12.12x + 6.15, R2 = 0.96. In the dose range between 5 and 13 Gy, the frequencies of cells with chromosomal aberrations did not differ significantly, i.e. the
2.2. Endpoints of germinating onion seeds The following endpoints of germinating onion roots were used as toxicity indicators: total chromosomal aberrations and different types of anaphase-telophase aberrations; micronuclei; and average length of germinating roots. For the cytogenetic study, roots were fixed in 96% ethyl alcohol: glacial acetic acid (3:1 v/v) immediately after exposure. After several days, the roots were stained with 1% acetic-orcein mixture (Medvedeva et al., 2014). Squashed slides were prepared from root tips, which were immediately analyzed by optical microscopy (Olympus CX31) at × 600 magnification and photographed. Cytogenetic abnormalities were studied using the method of Allium anaphase-telophase chromosome aberration assay and micronucleus test. The frequency of anaphase-telophase cells with chromosomal 2
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aberrations (data presented in the column “Other” of Table 1) and were less informative than the three major types of aberrations. In experiments carried out in 2017, in addition to analysis of chromosomal aberrations and the length of germinating seed roots, we conducted a micronucleus test of the apical meristem cells of germinating seed roots. The frequency of micronuclei in interphase cells of germinating seed roots exposed to a dose of γ-radiation of 1 Gy or higher was significantly different from the control (Fig. 1B). In the cells exposed to a low dose of 0.1 Gy, the frequency of micronuclei (1.2 ± 0.5%) was higher than in the control (0.6 ± 0.5%), but the difference was non-significant. That suggested lower sensitivity of the micronucleus test compared to the chromosomal assay for the cells of germinating onion seed roots exposed to low-dose radiation. The data in the dose-response curve for the micronuclei in the range between 0 and 4.5 Gy, can be described by the linear equation y = 3.27x+0.94, R2 = 0.99, as in the analysis of chromosomal aberrations. In the dose range between 2.6 and 13 Gy, the frequencies of cells with chromosomal aberrations did not differ, i.e. the dose–response curve exhibited a plateau. All data in the dose-response curve of the frequency of micronuclei can be approximated by the second-degree polynomial equation: y = – 0.24x2 + 3.50x + 1.27, R2 = 0.85 (Fig. 1B). The dose-response curves of the frequency of the micronuclei and the frequency of chromosomal aberrations had similar shapes, with an initial linear segment followed by a plateau, when there was no dose dependence. The plateau for the frequency of chromosomal aberrations was in the dose range between 5 and 13 Gy, while the plateau for the frequency of micronuclei was broader, extending from 2.6 to 13 Gy. At the highest dose, 13 Gy, the average frequency of micronuclei decreased, but it was not significantly different from the frequencies at lower doses (Fig. 1B). We performed correlation analysis to determine the relationship between the frequency of cells with micronuclei and the levels of different types of chromosomal aberrations (using the data of 2017 in Table 1). The frequency of micronuclei was significantly correlated to the levels of individual chromosomal aberrations such as fragments (r2 = 0.91 p < 0.01) and lagging chromosomes (r2 = 0.95 p < 0.01). When individual chromosomal aberrations were combined with the same types of abnormalities in the multiple aberrations, the frequency of micronuclei was significantly correlated only to the total level of fragments (r2 = 0.77 p < 0.05) but was not correlated to the total lagging chromosomes. Thus, the study of the chromosomal aberrations suggests that fragments are likely the main source of formation of micronuclei in cells of germinating seed roots.
Fig. 1. Absorbed dose (Gy) dependence of the frequencies of chromosomal aberrations (A) and micronuclei (B) in the cells of germinating onion seed roots (%). Approximation of the data with linear (1) and second-degree polynomial (2) models. Line (3) is the control. * – statistically significant difference from the control level (p < 0.05).
dose–response curve exhibited a plateau. All data in the dose-response curve of the frequency of chromosomal aberrations can be approximated by the second-degree polynomial equation: y = – 0.74x2 + 16.30x + 4.29, R2 = 0.98 (Fig. 1A). Three types of aberrations – bridges, fragments, and lagging chromosomes – made the major contribution to the total chromosomal aberrations (Table 1). Among these individual aberrations, the percentage of lagging chromosomes was somewhat higher (more than 20%), but at an exposure dose of 6.4 Gy or more, the percentages of lagging chromosomes and the bridges were virtually the same. The fragments constituted no more than 11%. As the exposure dose was raised, the frequencies of almost all types of chromosomal aberrations increased. However, at a dose of 5 Gy or higher, the percentage of fragments declined, reaching its minimum at the highest dose – 13 Gy (Table 1). In addition to these three types of individual aberrations, we detected multiple aberrations: two or three aberrations of the types mentioned above in a cell. At the highest exposure doses (6.4 and 13 Gy), the frequency of multiple aberrations was higher than the frequency of individual aberrations, reaching 28–59% (Table 1). An exposure dose of 2.6 Gy can be considered as the threshold of the occurrence of multiple aberrations: at this dose, multiple aberrations increased by an order of magnitude compared to aberrations at the dose of 1 Gy (from 0.9 to 9%). As the exposure dose was raised, an increase, similar to that observed in the main types of individual aberrations, was detected in the three types of multiple chromosomal aberrations. However, at doses of 3–4.5 Gy or higher, the percentage of fragments remained unchanged, in contrast to the other two types of aberrations (Table 1). In addition to the three major types of aberrations, we also detected chromosomal stickiness, multipolar and asymmetric mitoses, and ring chromosomes, but they constituted no more than 3% of the total
4. Discussion The standard Allium-test (with onion bulbs) has been traditionally used to assess chemical toxicity (Leme and Marin-Morales, 2009; Hoshina and Marin-Morales, 2009: Seth et al., 2008) while the toxicity of ionizing radiation has been studied far less extensively. A number of researchers (Cortés et al., 1990; Kumar et al., 2011; Perez-Talavera et., 2003; Sinovets et al., 2009; Vaijapurkar et al., 2001) reported that the standard Allium-test with bulbs can be effectively used to assess toxicity of ionizing radiation to such endpoints as root length, chromosomal aberrations, micronuclei, and mitotic index only at relatively high exposure doses (above 1.5–2 Gy). At low exposure doses (≤0.1 Gy), no inhibition of onion endpoints was observed, but two studies (Sinovets et al., 2009; Bolsunovsky et al., 2016b) detected stimulation of endpoints. As already noted, the Allium-test can be conducted with both bulbs and seeds. Moreover, seeds are more convenient test objects, as they are biologically dormant and genetically and physiologically uniform. Kovalchuk et al. (1998) reported a study in which onion seeds were used to assess genotoxicity of soil samples from the zone affected by the Chernobyl NPP. The authors found correlation between the total chromosomal aberrations in root cells of seedlings and content of 137Cs, 3
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frequency of micronuclei in the cells of germinating onion seed roots began in the range of 2.6–4.5 Gy, and that was consistent with the data reported for the micronuclei of the irradiated onion bulbs (George et al., 2014). The plateau for the frequency of the chromosomal aberrations in our experiments began at 5–6.4 Gy, i.e. close to the plateau for micronuclei (Fig. 1). At high exposure doses, however, we did not obtain another significant linear change in the frequency of micronuclei, which had been observed in the third phase in the study by George et al. (2014). Thus, we obtained a bi-phasic rather than a tri-phasic dose–response curve. In experiments with γ-irradiation of Allium sativum bulbs, George et al. (2014) obtained a quadri-phasic rather than a triphasic dose–response curve: between the dose-independent plateau and the second linear micronuclei frequency increase, there was a phase of a decrease in the frequency of micronuclei in the cells of Allium sativum roots. Our findings and the data reported in the study cited above suggested that different Allium species and different stages of development (bulbs and germinating seeds) of the same species (Allium cepa) showed dissimilar responses to γ-irradiation. In a study by Takatsuji et al. (2010), the authors exposed germinating onion seeds to three types of high energy accelerated heavy ions and observed micronuclei in the root tip cells. An increase in micronuclei induction was first recorded at 0.05 Gy, reaching its maximum at 1.0–1.5 Gy. In the dose range between 1 and 2 Gy, micronuclei induction either remained unchanged or decreased depending on the type of high energy heavy ions, i.e. bi- and tri-phasic dose–response curves were obtained. In the control, however, micronuclei induction was 0.4% (similar to our results), and at the highest exposure doses it reached 20–42%, depending on the type of high energy accelerated heavy ions. In that study, micronuclei induction was described with a dose response curve of an upward convex bell shape. The decrease in micronuclei frequency at high doses was caused by the lethal damage to cells done by heavy ions. The data discussed above show that the nonlinear dose dependence of cytogenetic abnormalities is usually characterized by a tri-phasic dose–response curve. However, some of the studies report bi-phasic dose–response curves (the present work and some of the curves in research by Takatsuji et al. (2010)) and quadri-phasic ones (George et al., 2014). Authors cited above (Geras'kin, 1995; Geras'kin et al., 2007; George et al., 2014) attribute the nonlinear dose dependence of cytogenetic abnormalities to differences in the status of DNA repair processes, with DNA repair activated by a certain threshold dose or level of DNA damage. At low doses, the effects of exposure to radiation are minor compared to spontaneous ones. Hence, the increase in micronuclei induction or chromosomal aberrations at the lower doses representing the first linear phase of the dose curve was associated with misrepair events. The following phase is plateau, when in spite of a considerable dose increase, no change is observed in the frequency of micronuclei or chromosomal aberrations. In that phase, as the dose is increased, repair mechanisms are activated in response to the radiation-induced DNA damage. As suggested by George et al. (2014), the DNA repair mechanisms may be activated by a threshold dose or level of DNA damage. In our experiments, 2.6 Gy may be the threshold dose activating DNA repair mechanisms, and this dose is similar to the threshold dose (2 Gy) reported by George et al. (2014). At the end of the plateau phase, as the dose is increased and the repair mechanisms become more effective, genetic damage may even be reduced. Usually, however, at a higher dose, the effectiveness of the repair systems decreases and, finally, they are suppressed, and the third phase of linear dose-dependent increase in aberrations is observed (George et al., 2014). As noted above, there are no available literature data on inhibition of the endpoints of onion (bulbs and seeds) by low doses of γ-radiation (≤0.1 Gy). The only study reporting irradiation of onion (Allium cepa) bulbs with γ-radiation (George et al., 2014) demonstrated that a significant increase in micronuclei induction relative to the control was only observed at the lowest dose, 0.7 Gy. That study on micronuclei
but they presented no data on absorbed doses of the seedling roots. The study also reported that the major aberrations contributing to the highest total percentage of chromosomal aberrations were bridges and fragments, which were the prevailing types of chromosomal aberrations in our experiments (Table 1). Tkalec et al. (2009) investigated the effect of electromagnetic fields (EMFs) on the cytogenetic and growth parameters of germinating onion seed roots. The authors showed that a more than tenfold increase in EMFs did not affect the length of the roots (like in our experiments), suggesting low sensitivity of physiological parameters of the roots to EMFs. However, the total chromosomal aberrations increased from 1 to 2% in the control to 4–7% in the treatment. EMFs induced such aberrations as lagging chromosomes, vagrants, disturbed anaphase, and chromosome stickiness. At the same time, the authors of that paper did not mention such aberrations as bridges and fragments. In our experiments, we irradiated germinating Allium cepa seed roots and obtained γ-radiation dose dependencies for such endpoints as the frequency of chromosomal aberrations and micronuclei. We have not found any data of other researchers on dose dependencies for the frequency of chromosomal aberrations after exposure of germinating onion seed roots to γ-radiation. There are, however, literature data on dose dependencies for the frequency of micronuclei after exposure of germinating onion seed roots to γ-radiation (Zhang et al., 2002, 2003; George et al., 2014) and high energy heavy ions (Takatsuji et al., 2010). The available data on radiation exposure of dry and wet seeds cannot be compared with the data on exposure of seedlings, as they exhibit different responses to radiation exposure. Rice (Oryza sativa, japonica) seeds were exposed to carbon ions at 20 Gy (Jin-Ming Shi et al., 2010), and that dose was lethal to the wet seeds and seedlings of rice while the dry seeds remained viable after the 20 Gy exposure. At a dose of 20 Gy, the frequency of chromosomal aberrations in root meristems of rice induced by carbon ion radiation was the highest for seedlings (22.5%) while for dry seeds and wet seeds it was considerably lower: 5.5 and 13%, respectively. The chromosomal aberrations in cells of rice seeds and seedlings included bridges, fragments, laggards, stickiness, and disturbed spindle. These types of chromosomal aberrations observed in cells of rice seedlings exposed to carbon ions are the same as the types of chromosomal aberrations in our experiments with germinating onion seed roots exposed to γ-radiation (Table 1). The difference in radiosensitivity between the dry and wet seeds may be caused by the difference in the water content. Roots of germinating seeds, as proliferating tissue meristems, have the highest sensitivity to the genotoxic effects of radiation factors. The data reported in a number of studies suggest the nonlinear dose dependence of the frequency of genetic disorders under exposure to low and medium doses of radiation. The authors of those studies (Geras'kin, 1995; Geras'kin et al., 2007; George et al., 2014) noted that the doseresponse curves showed not only a linear increase in genetic disorders but also a dose-independent part (a plateau). Such tri-phasic dose–response curves were obtained for various biological objects (Chinese hamster fibroblast cells and Vicia faba root cells, human lymphocytes, barley and onion root cells), suggesting that this behavior of the doseresponse curve could not be attributed to random experimental fluctuations but was rather caused by the similar responses of genetic structures to the dose increase. The dose-response curves obtained in the present study (Fig. 1) support the data cited above, which suggest nonlinear dose dependence of various genetic disorders in the region of low and medium exposure doses. The most noteworthy study was reported by George et al. (2014). The authors obtained nonlinear dependence of the frequency of micronuclei after irradiation of onion (Allium cepa) bulbs with γ-radiation. The linear increase in the micronuclei frequency in a range of doses between 0.7 Gy and 2 Gy was followed by a dose-independent plateau between 2 and 4 Gy, and then, at doses from 4 to 6 Gy, micronuclei induction increased again. In that study, the dose-independent response (plateau) began at 2 Gy. In our experiments, the plateau for the 4
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Acknowledgements
induction suggested that Allium cepa showed the lowest radiosensitivity among the 4 plant species studied. Similarly, in our experiments, the frequency of micronuclei in cells of germinating seed roots was significantly different from the control only at a dose of γ-radiation of 1 Gy or higher (Fig. 1B). However, in our experiments (Fig. 1A), the frequency of chromosomal aberrations showed the highest radiosensitivity to γ-radiation among the endpoints studied: at a low dose of 0.05 Gy or higher, it was different from the control. A similar conclusion, about the high radiosensitivity of the frequency of chromosomal aberrations to low-dose γ-radiation was made in a study by Geras'kin et al. (2007) for germinating spring barley (Hordeum vulgare) seeds. In that study, a triphasic dose–response curve was obtained for the frequency of chromosomal aberrations, and a significant increase in the frequency of chromosomal aberrations was observed at a dose of 0.05 Gy, like in the present study, for germinating onion seed roots (Fig. 1A). Chromosomal aberrations mentioned in the study by Geras'kin et al. (2007) were bridges, fragments, and lagging chromosomes. Lagging chromosomes made the largest contribution to the total chromosomal aberrations, and this is in good agreement with the results obtained in the present study (Table 1). That suggested high radiosensitivity of chromosomal assay compared to the micronucleus test for the cells of germinating onion seed roots exposed to low-dose radiation. The correlation analysis described above revealed significant positive correlation of the frequency of micronuclei to the levels of such chromosomal aberrations as fragments and, partly, lagging chromosomes in cells of germinating seed roots. Thus, the study of the chromosomal aberrations suggests that fragments are likely the main source of formation of micronuclei in cells of germinating seed roots. The review by Fenech et al. (2011) reported a study of the molecular mechanisms of micronucleus formation in human cells, which demonstrated that micronuclei can originate during anaphase from lagging acentric chromosomes or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Thus, in the interphase, the frequency of micronuclei is mainly determined by the frequency of chromosomal aberrations generated during anaphase. However, for plant cells, mechanisms of micronucleus formation remain insufficiently studied.
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5. Conclusion The main effects of γ-radiation observed in the root cells of germinating onion (Allium cepa) seeds include an increase in the inductions of chromosomal aberrations and micronuclei. The dose dependencies of the frequency of chromosomal aberrations and micronuclei are nonlinear: the initial linear part is followed by a plateau, where no dose dependency is observed. The dose-independent plateau for the frequency of micronuclei begins at 2.6 Gy, and the plateau for the frequency of chromosomal aberrations begins at 5 Gy. An increase in the frequency of the chromosomal aberrations was first observed after exposure to γ-radiation in the low-dose range, suggesting high radiosensitivity of chromosomal assay compared to the micronucleus test for the cells of germinating onion seed roots exposed to low-dose radiation. In the high-dose region of radiation (up to 13 Gy), the length of the roots after exposure did not significantly differ from the length of the germinating seed roots in the control. Correlation analysis revealed significant positive correlation of the micronuclei frequency to the frequencies of such chromosomal aberrations as fragments and, partly, lagging chromosomes. These findings clearly demonstrated the genetic effects of γ-radiation, low doses in particular, on germinating onion seed roots. Therefore, cytogenetic endpoints of germinating onion seed roots can be used to assess biological effects of low-dose γ-radiation. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. 5
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