Cytogenetic Effect of Low Dose γ-Radiation in Plant Test-Systems: NonLinear Dose-Effect Relationship
Stanislav Getas’kina*, Alla Oudalovaa, Vladimir Dikareva, Tatyana Evseevab, David
Copplestonec, Nina Dikarevaa, Elena ChernonogaaRussian Institute of Agricultural Radiology and Agroecology RAAS, 249020, Obninsk, Kaluga region,
Russia,b
Institute of Biology, Komi Scientific Center, Ural Division RAS, Kommunisticheskaya 28, 167982,
Syktyvkar, Russia,cEnvironment Agency, Warrington, U.K.

Over several decades, modelling the effects of ionizing radiation on biological system has reliedon the target principle [Timofeeff-Ressovsky et al., 1935], which assumes that cell damage ormodification to genes appear as a direct consequence of the exposure of biological macromolecules tocharged particles. It is assumed that there is no threshold for the induction of biological damage and thatthe effects observed are proportional to the energy absorbed. Following this principle, the average numberof hits per target should increase linearly with dose, and the yield of mutations per unit of dose is assumed
to be the same at both low and high doses. This principle has served as the scientific background for the
linear no-threshold (LNT) concept that forms the basis for the radiological protection for the public andthe environment [ICRP, 1990]. It follows from the LNT that there is an additional risk for human healthfrom exposure to any radiation level, even below natural background.
Since the mid 50s, however, the scientific basis for the LNT concept has been challenged as
experimental data have shown that, at low doses, there was a non-linear relationship in the dose response.
Luchnik and Timofeeff-Ressovsky were the first who showed a non-linear response to a low dose
exposure [Luchnik, 1957; Timofeeff-Ressovsky & Luchnik, 1960]. Since then, many data have been
accumulated which contradict the LNT model at low doses and dose rates. However, the hit-effectparadigm has become so strong and indissoluble that it has persisted even under the growing pressure ofscientific evidence for phenomena at low dose exposure that can not be successfully accounted for by the
LNT concept.
In recent years, additional information on non-targeted effects of radiation has been accumulated
following the first reports of an adaptive response in human lymphocytes [Olivieri et al., 1984] as well as
bystander mutagenic effect of alpha-particles [Nagasawa & Little, 1992; Mothersill et al., 1995]. Other
phenomena including genomic instability, low-dose hypersensitivity, and increased radiation resistanceeffects are also under study [Marples et al., 1997; Kadhim et al., 2004; Bonner, 2004].
The nonlinearity of the dose-effect relationship with low level exposures has been demonstrated in a
number of studies where chromosome aberrations were considered as the endpoint of interest. For
example, the number of radiation-induced dicentrics in human peripheral blood lymphocytes found in[Pohl-Ruling et al., 1983; Lloyd et al., 1988, 1992] did not exceed the control level at doses below 40
mGy, with some experimental points lying significantly below control values. Essential deviations ofchromosome aberrations appearance from linearity in mammals were also shown at higher doses of 100300 mGy [Luchnik & Sevankaev, 1976; Takahashi et al., 1982].
In other species, deviations of cytogenetic effect induced by low doses from linearity have also
been reported. For example, the dose response for cytogenetic effects in Chinese hamster fibroblasts andVicia faba germs at doses from 0 to 2.5 Gy was shown to be non linear with a plateau at low doses by[Zaichkina et al., 1992]. Frequency of chromosome aberrations in root meristem cells of Pisum sativum- 179 -

plantlets in the dose range of 0-10 Gy also showed non-linear responses with a plateau for doses up to 1
Gy [Zaka et al., 2002].
However the available information on dose-effect relationships at low doses for non-humanspecies is scarce despite its importance. In their natural environment, some non-human species may be at ahigher risk of impact than humans because of differences in ecological niches occupied [Geras’kin et. al.,2003]. Currently, radiation protection of the environment and maintenance of ecosystem sustainability isof a special concern and the development of a harmonized approach to human and biota protection has
been recognized as a challenge for modern radiobiology and radioecology [Copplestone et al., 2004;
Pentreath, 2002]. In this context, much more information on non-human species response to low level
exposures is needed.
This paper summarizes findings of several studies on the cytogenetic effects induced by low level
exposure to external γ-radiation in plant meristem cells.
Materials and Methods
Four of the experiments presented here were carried out on spring barley (Hordeum vulgare L.,
variety Zazerskiy 85) and one with Tradescantia (clone 02).
Barley is one of the most genetically well-studied crops and has been identified as an excellent
organism for studies of induced chromosome aberrations [Constantin & Nilan, 1982]. Barley has 14 (2n)relatively large (6-8 µm) chromosomes which are easy to identify. In addition, a general procedure forroot-tip aberration bio-assay is well-known, relatively quick and inexpensive to undertake. The following
protocols were assigned for treatment and exposure of barley seeds and germs:
Protocol I. Dry seeds of H. vulgare L. were acutely irradiated with 60Co γ-rays (in a “GammaCell”) at doses of 0.1, 0.5, 1, 5, 10, 25, 50, 100, 200, and 300 Gy. 24 h after exposure, the exposed seeds
were placed in Petri dishes on distilled water-moistened filter paper for germination. Sections of mainroots (approximately 5-10 mm long) were cut from 20-40 seedlings per dose point and fixed in acetic
alcohol (1:3).
Protocol II. Seeds of H. vulgare L. were soaked for 24 h in distilled water at +4 0С in the darkness
to synchronize cell division and provide evenness of swelling by beginning of germination [Konzak andNarayanan, 1977]. The seeds were removed from cold storage and maintained on moistened filter paper at
24 0С. After 12-16 h of germination, barley seedlings were irradiated with 137Сs γ-rays at doses of 10, 50,
100, 150, 200, 300, 500, 750 and 1000 mGy at a dose rate of 0.5 Gy/h (Lutch Irradiator, Latenergo,
Latvia). The germination continued until a root length of ≈ 10 mm was achieved and then 20-70 seedlings
per dose point were fixed as above.
Protocol III. Seeds of H. vulgare L. were kept for 24 h in distilled water at +4 °C to synchronize
cell division and exposed to 60Co γ-rays at doses of 3, 5, 10, 50, 100, 150, 250, and 300 mGy at a dose rateof 60 mGy/h immediately after removing from cold storage, then allowed to germinate at +24°C in Petridishes on filter paper wetted with distilled water. When the main root length reached about 10 mm, roottips of 20-80 seedlings per dose point were cut and fixed.
Protocol IV. Prior-exposure treatment of seeds was the same as in Protocol II. Doses of 5, 10, 50,
100, 150, 300, 500, 750, and 1000 mGy were delivered to seedlings 24 h after germination at +24°C at
three dose rates of 120, 300 and 900 mGy/h with 60Co γ-ray source (“Lutch Irradiator”, Latenergo, Latvia).
The germination continued until a root length of ≈ 10 mm was achieved. 15-50 seedlings per dose point
were fixed as above.
Preconditioning of seeds in cold storage provides a simultaneous initiation of barley seeds’
germination. So at the moment of irradiation, the cell population would be nearly homogeneous in termsof the cell cycle. Maximal frequency of scorable chromosome aberrations can then be registered in thefirst mitosis. However, it is not always possible to say precisely at what time cells come into mitosis as, in- 180 -

exposed cells, the cell cycle can be influenced not only by the treatment received (preconditioning,temperature, etc) but also by the type and severity of any impact (source of irradiation, dose rate, etc).
From published data [Sandhu et al., 1994], as well as from our pilot studies, the first peak of mitotic
activity in barley root tip cells was found at the moment when the main root reaches a length ofapproximately 10 mm, which was then taken as the point for samples to be fixed for cytogenetic analysis
in the barley studies.
In all four studies with H. vulgare L., temporary squash slides for cytogenetic analysis wereprepared from the root apical meristem of every seedling, coded and stained with aceto-orcein. In eachslide, all ana-telophase cells (from 700 to 9800 cells at dose point in various studies) were scored todetermine the fraction of cells with aberrations.
In the study with Tradescantia (2n=12), plants were grown under controlled conditions (t = 22 ± 1°С; 18 hours per day of illumination at 11580 Lux; 44% air humidity). Irradiation was started at a moment
when the first blossoming appeared in an inflorescence. At least 5 plants per dose point were exposed to γrays of 226Ra during 72 hours with various dose rates so that the resultant doses amounted to 0.2, 1.1, 2.5,
9.1, 22.2, 54.9, 91.0, 267.4, and 422.4 mGy, as measured at the inflorescence position. Different dose rates
were achieved by varying the distance of the plants from the source. Stamen hairs were analyzed from 4
up to 30 days of blossoming. Somatic mutations were registered according to [Ichikawa et al., 1978].
Results and discussion
1. Aberrant cells in barley root tips after seeds exposure (Protocol I)
Crops are an important component of food chains and have been extensively studied by the
Russian Institute of Agricultural Radiology and Agroecology (RIARAE). Numerous radiobiologicalstudies have been carried out in crops, including the study of the effect of ionizing radiation on
reproduction potential of seeds (pre and post sowing) [Alexakhin & Korneyev, 1992]. With this historicalbackground and the well-established techniques in RIARAE for seed irradiation, acute γ-exposure of seeds
was used to study cytogenetic effects in crops induced by low and moderate doses to investigate theapplication of the LNT concept in non-human species [Geras’kin et al., 1993; 1995; 1997a].
Generally, the range of plants’ radioresistance is higher than that of mammals, with dormant seeds
being more radioresistant than during crop vegetation [Sarapultzev & Geras’kin, 1993]. The dose range inProtocol I was chosen to compare the plants radiosensitivity for different stages of ontogenesis. For
example, it is known that, for Monocotyledons, radioresistance of dormant seeds is 4.8-5.6 times higher
than that for vegetating plants [Sarapultzev & Geras’kin, 1993]. Thus, the doses from 0.1 to 300 Gy
chosen for exposure of barley dry seeds could be considered as small (up to 5-10 Gy) and moderate dosevalues, regarding to high radioresistance of seeds.
Fig. 1 shows the frequency of aberrant cells (AC) observed in the root meristem of barleyseedlings following exposure of the dormant seeds [Geras’kin et al., 1997a]. There is no significant
difference from the control level following exposure to doses of 0.1 and 0.5 Gy. However at higher doses,
the AC frequency is elevated compared with an unirradiated control, but the relationship shows nodependence on exposure applied in a range from 1 to 25 Gy, despite the 10 times increase in absorbed
dose. Only at doses above 25 Gy is there an apparent linear increase in cytogenetic damage withincreasing radiation dose.
Despite all the criticism directed towards the LNT concept, the fundamental principles regarding
the interaction of radiation with DNA and other biological macromolecules is known to induce primarydamage. Thus the effect of ionizing radiation at a molecular level is actually non-threshold as the energyof any charged particle is far above the binding energy in biological macromolecules. Consequently, adose is specified as being low if a critical target receives not more than one radiation ‘hit’ [Kellerer, 1976].
Being aware of the critical target volume (i.e. size of cell or cell nucleus), it is possible to determine the- 181 -

Aberrant cells, %

12

8

4

0
0.0

0.1

1.0

10.0

100.0

Dose, Gy

Figure 1. Frequency of aberrant cells (mean ± s.e.) in barley root meristem in dependence on doseabsorbed by dormant seeds.
upper margin of low dose range based on the stochastic theory of radiation track distribution in a cell
population [Spitkovskij et al., 1994b; Oudalova et al., 2002]. At doses from natural background up to thisupper limit of the low dose region, the mean energy deposited in a cell’s sensitive volume is a constant;with discordances from ‘one-track’ events being negligibly small. As the dose increases within this range,
only the fraction of targets experiencing the radiation-absorption event increases linearly but not theenergy absorbed in a single affected target volume [Bond et al., 1988]. Therefore, there seems to be noreason for an induction of response patterns different from background effect appearance, and an AC
frequency should not differ significantly from the spontaneously observed level. This consideration
underlies the first part of the dose-response curve in Fig. 1, i.e. between the control and 1 Gy, where there
is no increase of the AC frequency over the control level.
At doses from 1 to 25 Gy, a ‘plateau’ in dose curve is observed. A dose-independence of
cytogenetic effect in a low dose region of dose curves, ‘plateau’, has been reported in many studies, inparticular, for human peripheral blood lymphocytes and melanoma cells, Chinese hamster fibroblasts,
Vicia faba and Pisum sativum seedlings [Sevankaev, 1991; Shmakova et al., 2002; Zaichkina et al., 1992,
Zaka et al., 2002]. To explain this deviation from linearity, most authors propose a hypothesis onradiation-induced repair system, which is triggered by a certain dose or certain level of biological damage[Luchnik & Sevankaev, 1976; Geras’kin, 1995a; Zaka et al., 2002]. In [Geras’kin, 1995b], induction of themutagenic SOS-response, which is normally repressed, was suggested as providing restoration of cell
survival but at the cost of an elevated frequency of genetic defects. At low doses, the contribution fromradiation-induced effects compared with authentic spontaneous ones is rather small [Sarapultzev &Geras’kin, 1993; Pollycove & Feinendegen, 2001a] and so an increase in cytogenetic damage throughmisrepair appears important. The observation of the “plateau” in the dose dependency is a sign of theactivation of repair mechanisms in response to the exposure received. These could be registered throughalterations in other response patterns such as chromatin structure transformation or modifications in gene
expression [Spitkovskij et al., 1994a]. Within the plateau range, the occurrence of scorable chromosome
abnormalities reflects complex cellular responses that are triggered by the external insult (i.e. radiation
exposure) but they do not depend on its value. As a result, an anomalous dose range is observed
experimentally within which 2-10 fold changes in dose is not accompanied by any significant increase inchromosome aberration frequencies. As the dose increases further, the potential of the SOS-response
systems to respond effectively decreases until it is overwhelmed and the dose-response curve then shows a- 182 -

continuous increase in cytogenetic effect with increasing dose.
2. Aberrant cells in root tips after germs exposure (Protocol II)
To reduce misinterpretation and uncertainty resulting from the influence of physiologicalprocesses which may occur during germination after seeds exposure to ionising radiation and prior toscoring of chromosome aberrations in seedlings, other experimental protocols were designed (Protocols IIIV) to expose germinated and not dormant seeds. Actively dividing cells of meristem tissues are the mostradiosensitive parts of a plant. Previous work has demonstrated that the test-system of “AC frequency inthe intercalary meristem of spring barley” is as sensitive to γ-radiation as the generally accepted test of“aberrations in lymphocytes of human peripheral blood” [Geras’kin et al., 1996].
The results of cytogenetic analysis in barley root meristem after irradiation of 12-16 h seedlings(Protocol II) at doses from 10 mGy to 1 Gy are presented in Fig. 2 [Geras’kin et al., 1999]. The
relationship between AC frequency and dose is obviously non-linear. A significant difference in AC
frequency from the control was found at doses > 50 mGy (p<5%, Kolmogoroff-Smirnov test [Sachs,
1976]). There are three parts to the dose curve which essentially describe differences in the dependencesof the cytogenetic disturbance on dose. Thus, at doses from 500 mGy and higher, the AC percentage
significantly differs from the control level (p<1%) and increases with increasing dose. However, within
the dose range 50-750 mGy there are no significant differences in the AC occurrence, although the effectobserved is higher than the spontaneous level of the AC frequency (p<5%). Hence, a ‘plateau’ in thisstudy is revealed, and a non-linear relationship in dose-cytogenetic effect dependence is observed inbarley root tip cells.

3

Aberrant cells, %

4

3

2

2

1

0
0

200

400

600

800

1000

Dose, mGy
Figure 2. Frequency of aberrant cells (mean ± s.e.) in barley root meristem in dependence on doseabsorbed by 12-16 h seedlings and approximation of the data with linear (1), piecewise linear (2) and 4th
degree polynomial (3) models.
The use of the linear extrapolation from high doses into the low-dose region for an estimation ofhealth risk and, correspondingly, in radiological protection, has been justified by a lack of reliable data,
which are increasingly difficult to obtain at lower exposures because of large sample size needed [Upton,
2003; Brenner et al., 2003; Bonner, 2004; Tubiana, 1998]. At present, many authors have expressed theopinion that the validity of the LNT concept needs to be tested [Bonner, 2004; Tubiana, 1998, Mothersill& Seymour, 2004]. Under the increasing weight of experimental evidence which supports the non- 183 -linearity at low doses, there has been a shift in a perception of the LNT hypothesis from the indisputably
correct dogma to one which is the most convenient, prudent and transparent operational tool inradioprotection and radiotherapy practise [Tubiana, 1998; Mothersill & Seymour, 2004]. In the interim, it
is useful to test the application of the LNT concept and to continue to challenge its superiority from not
only biological but also a mathematical viewpoint.
With this in mind, a comparison of goodness-of-fit of AC yields versus radiation dose using
mathematical models of different types and complexities was performed on an example of the dataobtained under Protocol II [Geras’kin et al., 1999; Oudalova et al., 2005]. A set of polynomial models( ym =m∑ a ⋅ x ) including linear function as a special case (m = 1), and a piecewise-linear (PL) model
i

i =0

i

were used. The PL model pre-supposes a non-linearity of a dose dependency, which consists of several
linear parts with different slopes, including a dose-independent plateau in a dose range [D1, D2], and has
five free parameters.
For the polynomial models, values of free parameters were found from generalized linear
regression [Draper & Smith, 1981]. In particular, an intercept and slope of the linear model (y = a + b·x)
are a = (0.74 ± 0.21) %, b = (3.00 ± 0.46) 10-3 %/mGy, correspondingly. Free parameters of the PL model
were found using an iterative regression tool based on the method of coordinatewise descent [Vasiliev,
1988]. Important findings from the PL model verification are the lower and upper limits of the ‘plateau’that are defined as D1 = 83.4 mGy and D2 = 513.7 mGy for the experimental data obtained in this study.
Fitting the data on the AC frequency in barley root meristem with the linear and PL models is illustrated inFig. 2.
The various models applied for the data approximation were followed by a quality review of theseapproximations with several different criteria, and the results are shown in Table 1. All six models are able
to fit the data satisfactorily (F not less than 12.5, p<5%). However, the polynomial models of 2, 3, 4
degrees and the PL model show the lower values of residual sum of squires, SSres, than the linear model,
resulting in higher Fisher statistics, F, and multiple correlation coefficient, R2 (Table 1). It is known thatthe predictive reliability of a model breaks down as the number of free parameters increases [Algorithms...,1988]. To test the relative complexity of the models, the criteria of structural identification, T [Geras’kin& Sarapult’zev, 1993] was used. This method penalizes a model for the more additional free parameters it
has; so that the lower T-value the more optimum the ratio between the complexity and goodness of data
fitting the model has. From Table 1, the lowest value of the T-criteria is acquired by the polynomial modelof 4th degree as well as PL model despite their 5 free parameters (np=5) and its correspondingly highcomplexity. This result means that the improved quality of approximation is reached not so much bymaking the model more complex, but due to its more adequate mathematical description (or, in otherwords, functional isomorphism) of the biological phenomenon.
To check the significance of the approximation improvement, a hypothesis was tested whether the
linear model fits the experimental data significantly worse than other models, using the Hayek criterion, H,[Gofman, 1990]. From the calculated values of H (Table 1), the goodness’s of experimental data fit byboth the PL model and 4th degree polynom is significantly higher than by the linear model (H>H0.95=1.96).
A common feature for two the best functions is a tendency to fit a ‘plateau’ in the cytogenetic
disturbances occurrence (Fig. 2). The 4th-degree polynom is, however, consistent with the biologicalresponse observed only in the dose range studied, 0 – 1 Gy, while at higher doses this function drops to ∞ very fast, which illustrates an errancy of formalistic approach to data verification. On contrary, the good
fit with the PL model is provided through conformity achieved between a biological phenomenon and its
mathematical model. Consequently, this study shows that the PL model which assumes non-linearity ofthe dose-effect dependency, and implies the presence of a plateau, fits the cytogenetic disturbances- 184 -

occurrence in barley root meristem cells within the low dose range significantly better than any otheramong the tested models (and, in particular, better than the linear approach).
Table 1. Comparison of approximation quality of aberrant cells frequencies with different models. Data
are obtained in dose range 0-1000 mGy (Protocol II) and 0-300 mGy (Protocol III)Modelnp
0-1000 mGy
0-300 mGy
SSR F
R2 , % T
H
SSR F
R2 , % T
H
1. Linear
2 1.80 41.7 83.9 0.45 0.41 24.4 77.7 0.12 2. Piecewise linear
5 0.30 180.2 97.3 0.30 3.55*
0.03 228.9 98.3 0.04 4.92**
3. Polynom (m= 2)
3 1.69 39.3 84.9 0.72 0.50
0.24 40.7 87.1 0.12 1.53
4. Polynomial (m= 3) 4 1.20 49.7 89.2 0.80 1.25
0.06 161.5 97.0 0.05 4.04**
5. Polynomial (m= 4) 5 0.21 258.8 98.1 0.21 4.35** 0.02 377.5 99.0 0.02 6.38**
6. Polynomial (m= 5) 6 2.70 12.5 75.8 4.06
0.01 459.5 99.4 0.02 7.09**
7. Piecewise linear II 3
0.11 91.3 93.8 0.06 2.84*** – α<1%, * – α<5% - model fits the data significantly better than the linear model;

Aberrant cells frequency, %3. Aberrant cells in root tips after germs exposure (Protocol III)The study of AC frequencies induced by low doses up to 300 mGy (Protocol III) was initiated toget more information on dose-cytogenetic effect relationship at doses below 50 mGy. The lack of suchinformation became obvious at analysis and modeling of the previous experiment data at doses below 100
mGy, as there were only two experimental point below 83.4 mGy, the plateau lowest limit (Fig. 2).

3

2.5

2.0

1

1.5

2

1.0

0.5
0

100

200

300

Dose, mGy
Figure 3. Frequency of aberrant cells (mean ± s.e.) in barley root meristem in dependence on doseabsorbed by germs (Protocol III) and approximation of the data with linear (1), piecewise linear (2), andpolynomial of 5th degree (3) models.
The common difficulty in assessing effects observed at low level exposures and making statisticalinferences is related to the lack of a suitable quantity of available data to overcome uncertainties resulting
from “experimental error” or “inter-species variation” when the induced effect itself has a small
magnitude. In an attempt to provide a lower uncertainty of AC frequency estimates, exposure in thisexperiment was delivered to initiated barley germs immediately after their removal from cold storage,- 185 -

assuming that in this case a cell population would be found in the most synchronized state. Moreover,attempts were made to provide experimental volumes that were as large as reasonably possible. Thus,
2000-9800 ana-telophase cells were scored at different doses as opposite to 1300-2600 cells in theprevious study.
Data from AC scoring are presented in Fig. 3. Significant increase of cytogenetic damage over thecontrol level is found at a γ-rays exposure of > 100 mGy as follows from Student t-test, or > 50 mGy as
follows from non-parametric Kolmogorov-Smirnov test (p<5%), which is in a good accordance with theprevious study. There are no significant changes in AC occurrence within the dose range from 3 to 100
mGy. AC frequencies at doses of 100, 150 and 250 mGy are significantly different to the control level, but
do not differ from each other. The yield of cytogenetic damage at 300 mGy increases significantly overthe values obtained at all other doses, even 250 mGy (p<5%).
To test a validity of the LNT extrapolation, a goodness-of-fit of the data on the AC frequency
approximation by different models was performed. A set of polynomial models, and several piecewiselinear models of different shape were used. The results are presented in Table 1. Piecewise linear model II
assumes an absence of dose dependence below a threshold dose (D0) and a linear dose-dependence at
higher doses. All models were able to fit the data satisfactorily (F not less than 24, p<0.1%). However thebest models (Table 1) were the polynomial 5- (Fig. 3, curve 3) and 4-degree functions as well as the PL
model that includes a ‘plateau’ (Fig. 3, curve 2). A common feature for these three best functions is anabsence (for the PL model) or very slight dependence (for the polynomial models) of AC yield on dose at
low doses, as can be seen from plotting the best-of-fit functions (Fig. 3). The linear function shows theworst characteristics of the data fitting (Table 1) obtained in this study.
4. Dose-cytogenetic effect relationship at different dose rates (Protocol IV)
It is well understood that the dose rate is of importance in assessing radiation-induced effects andthe risks at low doses. Numerous studies have reported direct dose-rate effects, with an absence of dosedependence and even an inverse dose-rate effect being found as well [Lyon et al., 1972; Sorensen et al.,
2000; Min et al., 2003]. In the LNT concept, the risk of detrimental effects per unit absorbed dose issmaller for lower doses than at higher exposures, which is acknowledged by an application of the doserate effectiveness factor [Tubiana, 1998; Sorensen et al., 2000]. In territories contaminated after severeradiation accidents, both humans and the biota have been exposed to radiation at dose rates that varies
through time and space, and the doses absorbed during the cellular and ontogenetic cycles of development
are consequently a function of the dose rate. Therefore, an issue of varying dose rate is of high relevancein regard to radiological regulation and practice policy.
To study an effect of dose rate value on cytogenetic damage level, barley germs were exposed atthree dose rates of 120, 300 and 900 mGy/h (Protocol IV) and ACs were scored in seedling root meristem[Oudalova et al., 2002]. Frequencies of ACs obtained in this study are presented in Fig. 4. Dosedependences at different dose rates keep the shape with a characteristic ‘plateau’ region of 10-300 mGy,and can be approximated with the PL model much better then with the linear one. There is a change inmagnitude of the AC yield with a change of dose rate used. Remarkably, in the exposure range
investigated, the induced cytogenetical disturbances appeared in a reverse dependence on dose rate, andthe lower the dose rate the higher the AC frequency (Fig. 4). Significant increase over the control (p<5%)
was found from dose of 10, 100, and 500 mGy and above for dose rates of 120, 300 and 900 mGy/h,correspondingly.
Observations of a reversed dependency of mutation frequency [Lyon et al., 1972] and DNA
damage [Min et al., 2003] on dose rate at low-level exposures are known. For example, more point
mutations and chromosome aberrations per unit dose at low level radiation than at higher levels were
found in [Shevchenko et al., 1972]. The most generalised explanation refers to an assumption of a
- 186 -

diminished activation of repair at very low dose rates [Min et al., 2003]. In other words, the inducible
repair system is probably activated by a certain threshold value of damage [Shevchenko et al., 1972;
Calkins, 1973]. This means that, at low exposures, the accumulation of damage needs to reach a certain
value or threshold before the repair mechanism is switched on. The lower the intensity of impact (i.e. doserate) the more damage is required, which results in an inverse dependence of AC registered on dose rate inthis exposure range.
3.0

Aberrant cells, %

2.5

2.0

1

1.5

2
1.0

3
0.5
0.1

1.0

10.0

100.0

1000.0Dose, mGy

Figure 4. Frequency of aberrant cells (mean ± s.e.) in barley root meristem in dependence on doseabsorbed by 24 h seedlings at dose rate of 120 mGy/h (1), 300 mGy/h (2), and 900 mGy/h (3).
An important finding of this study is that a non-linear shape of dose-effect dependence withplateau in low dose region is confirmed, and the independence of dose curve profile on dose rate in thestudied exposure range is shown. An inverse dependence of chromosome aberration yield and dose rate isdemonstrated for the first time in plant meristem cells, and requires further investigation. To obtain more
information on the role of dose rate in cytogenetical damage induction patterns, a study identical to thework done under Protocol III has been launched but with several different dose rates.
5. Somatic mutations in Tradescantia stamen hair cells
To corroborate finding obtained with another plant system, Tradescantia (clone 02) was chosen as
test-species in next experiment [Evseeva & Geras’kin, 2001]. Tradescantia has known as the best object
for genetic and radiobiological studies [Sax, 1938; Ma et al., 1996] and been useful for detection of thegenetic effects of both ionizing radiations and chemical mutagens at low exposure levels [Ichikawa, 1992].
The Tradescantia-Stamen-Hair-Mutation (Trad-SHM) bioassay uses the mitotic cells of stamen hairs formutation induction in which the stamen hair cells mutate from dominant blue to pink constitutes a system
unique in sensitivity; it can register doses of X-rays as low as several mGy [Sparrow et al., 1972].
Exposures in the study of radiation-induced somatic mutations in Tradescantia stamen hair cells[Evseeva & Geras’kin, 2001] were planned to provide detailed information at very low doses by choosing
a dose interval between experimental points that was as small as possible. Experimental dependency ofsomatic mutation frequency on dose value presented in Fig. 5 demonstrates an obvious non-monotonousshape showing common features with dose dependencies obtained in barley studies (Fig. 1-4). Three dose
ranges could be separated. At the exposure to doses of up to 9.1 mGy, the registered frequencies of- 187 -

Somatic mutations, %

1.0

0.8

0.6

0.4

0.2

0.0
0

1

10

100

1000

Dose, mGy
Figure 5. Frequency of somatic mutations (mean ± s.e.) in stamen hair cells from γ-ray-exposed
Tradescantia plants
mutations do not differ from the level of spontaneous disturbances and are not significantly different fromthe control. In the second dose range, namely at doses of 22.2, 54.9 and 91.0 mGy, occurrence of somatic
mutations is significantly higher than the control level and the values observed at the lower doses (up to 10mGy) but does not significantly change within this dose range, which indicates a ‘plateau’. When the dose
increases over 100 mGy, a steady increase of the mutation frequency is observed; somatic mutation
occurrence at doses of 267.4 and 422.4 mGy is significantly over the previous values at 0-9.1 mGy and9.1-91.0 mGy.
Conclusion
In the present study, attempts were made to investigate the biological effect of ionising radiation atlevels similar to those that may be observed in the environment. A correct assessment of risk of low doses
for human health is impossible on a basis of current epidemiological data because of large uncertainties
and experimental difficulties. There is a need to develop a more comprehensive understanding of theprocesses which govern cellular responses at low level exposures to ionising radiation. These processes
are likely to have a lot in common, at least for eukaryotic organisms, because of uniformity infundamental principles of genome organization and functioning. It is becoming increasingly clear that at
low doses indirect and non-targeted effects play a crucial role in cell response that are, in some ways,
similar to systematic stress [Mothersill & Seymour, 2004; Kadhim et al., 2004] and relevant toenvironmental toxins other than radiation. In this context, high sensitivity, low level of spontaneous
mutagenesis, accessibility to large volumes of compatible experimental data, and acknowledgement inenvironmental genotoxins testing make those plant-based systems excellent models for studying low-dose
effects.
The non-linearity and presence of plateaus in dose dependencies for cytogenetic disturbances havebeen reported in a number of works carried out with different species and test-systems. On the basis ofthese information and data obtained in several studies on cytogenetic effects in plant meristem, a conceptof biological effect of low-level radiation on cell was suggested [Geras’kin, 1995b; Geras’kin et al.,1997b]. It postulates uniformity for different species relating to an essentially non-linear dose dependence
shape, with only species-specific variations in critical doses at which slope modifications appear and thatthese critical doses depend on species radiosensitivity. The results of several studies presented in thisreview confirm the main postulates of this concept [Geras’kin, 1995b]. For example, the ‘plateau’ limits in- 188 -

dose curve observed by the Trad-SHM bioassay, 20-200 mGy (Fig. 5), are shifted down to smaller doses
in comparison to 80-500 mGy for barley (Fig. 2), which is in a perfect accordance with a higher
radiosensitivity of Tradescantia stamen hairs than that of barley cells. Furthermore, the concept[Geras’kin, 1995b] supposes an existence of one more dose-independent part that should not differ fromthe level of spontaneous cytogenetic variability. With the Trad-SHM bioassay, this theoretical prediction
has, for the first time, received an empirical confirmation on vegetative object. Indeed, the high sensitivity
and low level of spontaneous mutagenesis in Tradescantia made it possible to reveal such a dose range as
low as 0.2 – 9.1 mGy (Fig. 5).
Ignoring the fact that the non-linear character of dose−response curves leads to a substantial
underestimation of cancer risks, if high doses and linear dose−response curves are used for estimation of
the hazard of low-level radiation, the following points can be made:• Although many researchers believe that the estimation of cancer risks by the linear extrapolation ofhigh-dose data to the range of low radiation doses may only give overestimated values [Tubiana,
1998], the data presented in this paper suggest that the LNT concept should not be used for estimating
the risks of genetic defects induced by low-level radiation, since this concept has no sound biologicalunderpinning and comes into contradiction with available experimental and epidemiological data
[Gofman, 1990; Pollycove & Feinendegen, 2001b; Wei et al., 1990].
• The linear model is presumed to be advantageous as simple and prudent at modeling ambiguous
biological effects at low doses, so its benefits follow from mathematical properties of the linear
function, while the biological background of the LNT has been admitted as controversial. Findings ofthis work demonstrate, however, that it is not hard to call in question advantages of the LNT in fitting
available experimental data from mathematics point of view, as well.
• New data obtained in these studies concern a perception of fundamental mechanisms governing cell
response to low level exposures. These findings are of general biological interest, since response to
low level exposures is one of the manifestations of basic laws determining and ensuring the living
systems resistance and their adaptation potential under varying habitat conditions. An accumulation of
information on cell responses to low level exposures and validating LNT hypothesis eligibility are
relevant not only to an improvement of radiation safety standards but to the development of a general
ideology for the public and environment protection. It should be acknowledged that more studies areneeded for further validation of concepts for risk assessment at environmentally relevant levels.
Acknowledgements
Russian Foundation for Basic Research (contract № 03-04-96353), ISTC project № 3003 and INTAS(award Ref. Nr. 04-83-2796) have contributed to funding studies presented in this paper.
References
Alexakhin, R.M. & Korneyev, N.A. (eds). (1992) Agricultural Radioecology. Ecologiya Publish., Moscow (inRussian).
Algorithms and Programmes for Dependencies Reconstruction (1984) Ed. V.N. Vapnik, Nauka Publish., Moscow (inRussian).
Bond, V.P., Feinendegen, L.E., Booz, J. (1988) What is a ‘low dose’ of radiation? Int. J. Radiat. Biol., 53(1), 1-12.
Bonner, W.M. (2004) Phenomena leading to cell survival values which deviate from linear-quadratic models, Mutat.
Res., 568, 33-39.
Brenner, D.J., Doll, R., Googhead, D.T., Hall, E.J., Land, C.E., Little, J.B., Lubin, J.H., Preston, D.L., Preston, R.J.,
Puskin, J.S., Ron., E., Sachs, R.K., Samet, J.M., Setlow, R.B., Zaider M. (2003) Cancer risk attributable tolow doses of ionising radiation: assessing what we really know, Proc. Natl. Acad.Sci. U.S.A., 100, 1376113766.

- 189 -

Calkins, G. (1973) The T-N-PR Model Radiation Response. J. Theor. Biol., 39, 609-622.
Constantin, M.J. & Nilan, R.A. (1982) Chromosome aberration assays in barley (Hordeum vulgare). A report of the
U.S. Environmental Protection Agency Gen-Tox Program, Mutat. Res., 99, 13-36.
Copplestone, D., Howard, B.J., Bréchignac, F. (2004) The ecological relevance of current environmental protection
from exposure to ionising radiation. J. Environm. Radioact., 74, 31-41.
Draper, N.R. & Smith, H. (1981) Applied Regression Analysis, J. Wiley & Sons, New York.
Evseeva, T.I. & Geras’kin, S.A. (2001) Radioactive and Non-Radioactive Factors Combined Effect on Tradescantia.
Ural Division RAS Publishers, Ekaterinbourg. (in Russian)Geras’kin, S.A. & Sarapul’tzev, B.I. (1993) Automatic classification of biological objects on the level of theirradioresistence, Automat. Rem. Contr., 54, 182-189.
Geras’kin, S.A., Zjablitzkaja, Ye.Ya., Oudalova, A.A. (1993) Statistical analysis of mutafacient efficiency of low
doses chronic irradiation on technogenic pollution background. Russ. J. Genet., 9(11), 1901-1913.
Geras’kin, S.A. (1995a) Critical survey of modern concepts and approaches to the low doses of ionising radiation
biological effect estimation, Radiat.Biol. Radioekol., 35(5), 563-571.
Geras’kin, S.A. (1995b) Concept of biological effect of low dose radiation on cells, Radiat.Biol. Radioekol., 35(5),571-580.
Geras’kin, S.A., Zyablitskaya, Ye.Ya., Udalova, A.A. (1995) Radiation-induced structural mutations in root
meristem of γ-irradiated hexaploid wheat seeds. Radiat.Biol. Radioekol., 35(2), 137-149.
Geras’kin, S.A., Dikarev, V.G., Dikareva, N.S., Oudalova, A.A. (1996) Effect of ionising irradiation or heavy metalson the frequency of chromosome aberrations in spring barley leaf meristem, Russ. J.Genetics, 32(2), 240245.
Geras’kin, S.A., Zjablitzkaja, Ye.Ya., Oudalova, A.A., Sharapova, I.V. (1997a) Structural mutations in the root
meristem of irradiated barley seeds. Radiat.Biol. Radioekol., 37(1), 82-90.
Geras'kin, S.A., Dikarev, V.G., Oudalova, A.A., Alexakhin, R.M. (1997b) Problem of Correct Assessment of
Cytogenetic Effect Induced by Low Doses of Ionising Radiation. Proceedings of Conference - Low Doses of
Ionising Radiation: Biological Effects and Regulatory Control. Seville, 1997. IAEA-CN-67/205, 687-690.
Geras’kin, S.A., Dikarev, V.G., Oudalova, A.A., Dikareva, N.S. (1999) Cytogenetic damages in root meristem ofbarley seedlings after low dose irradiation, Radiat. Biol. Radioekol., 39, 373-383.
Geras’kin, S.A., Kim, J.K., Fesenko, S.V., Dikarev, V.G., Oudalova, A.A. (2003) Cytogenetic effects of low dose
ionizing radiation in plants and a problem of the radiological protection of the environment. Proceedings ofthe International conference on the Protection of the Environment from the Effects of Ionizing Radiation.
Stockholm, Sweden, 2003. IAEA-CN-109, 272-275.
Gofman, J. (1990) Radiation-Induced Cancer from Low Dose Exposure: an Independent Analysis, CNR Book
Division, San Francisco.
Ichikawa, S., Nauman, C.H., Sparrow, A.H., Takahashi, C.S. (1978) Influence of radiation exposure rate on somatic
mutation frequency and loss of reproductive integrity in tradescantia stamen hairs, Mutat. Res., 52, 171-180.
Ichikawa, S. (1992) Tradescantia stamen-hair system as an excellent botanical tester of mutagenicity: its responses
to ionizing radiations and chemical mutagens, and some synergistic effects found, Mutat. Res., 270, 3-22
ICRP (1990) Recommendations of the International Commission on Radiological Protection. ICRP Publication 60,
Annals of the ICRP, 21, 1-3.
Kadhim, M.A., Moore, S.R., Goodwin, E.H. (2004) Interrelationships amongst radiation-induced genomic instability,
bystander effects, and the adaptive response, Mutat. Res., 568, 21-32.
Kellerer, A.M. (1976) A Survey of Approaches to Radiation Biophysics. Proceedings of the Fifth Symposium on
Microdosimetry, Luxembourg: Commission of the European Communities, EUR 5452, edited by J. Booz,
H.G. Ebert and B.G.R. Smith, 409-442.
Konzak, C.F. & Narayanan, K.R. (1977) In: Manual on Mutation Breeding (Second Edition), FAO/IAEA Tech. Rep.,
Series No. 119, IAEA, Vienna, 71-74.

- 190 -

Lloyd, D.C., Edvards, A.A., Leonard, A., Deknut, G.L., Natarajan, A.T., Obe, G., Palitti, F., Tanzarella, A., Tawn,
E.J. (1988) Frequencies of chromosomal aberrations induced in human blood lymphocytes by low doses of
X-rays, Int. J. Radiat. Biol., 53, 49-55.
Lloyd, D.C., Edvards, A.A., Leonard, A., Deknut, G.L., Verschaeve, L., Natarajan, A.T., Darrudi, F., Obe, G., Palitti,F., Tanzarella, A., Tawn, E.J. (1992) Chromosomal aberrations in human lymphocytes induced in vitro byvery low doses of X-rays, Int. J. Radiat. Biol., 61, 335-343.
Lyon, M.F., Papworth, D.C., Phillips, R.J.S. (1972) Dose-Rate and Mutation Frequency after Irradiation of Mouse
Spermatogonia. Nature. New Biol., 238(82), 101-104.
Luchnik, N.V. (1957) About anomaly reaction at low doses of radiation, Biophyzica, 2, 86-93.
Luchnik, N.V. & Sevankaev, A.V. (1976) Radiation-induced chromosomal aberrations in human lymphocytes. 1.
Dependence on the dose of gamma-rays and on anomaly of low doses, Mutat. Res., 36, 363-378.
Ma, T.H., Xu, C., Liao, S., McConnell, H., Jeong, B.S., Won, C.D. (1996) In situ monitoring with the Tradescantia
bioassays on the genotoxicity of gaseous emissions from a closed landfill site and an incinerator, Mutat.
Res., 359(1), 39-52.
Marples, B., Lambin, P., Skov, K.A., Joiner, M.C. (1997) Low dose hyper-radiosensitivity and increased
radioresistance in mammalian cells, Int. J. Radiat. Biol., 71, 721-735.
Min, J., Lee, C.W., Gu, M.B. (2003) Gamma-radiation dose-rate effects on DNA damage and toxicity in bacterial
cells, Radiat. Environ. Biophys., 42, 189-192.
Mothersill, C. & Seymour, C. (2004) Radiation-induced bystander effects and adaptive responses—the Yin and Yangof low dose radiobiology? Mutat. Res., 568, 121-128.
Mothersill, C., Harney, J., Lyng, F., Cottell, D., Parsons, K., Murphy, D., Seymour, C.B. (1995) Primary explants ofhuman uroepithelium show an unusual response to low-dose irradiation with cobalt-60 gamma rays, Radiat.
Res., 142, 181-187.
Nagasawa, H., & Little, J.B, (1992) Induction of sister chromatid exchanges by extremely low doses of alfa-particles,
Cancer Res., 52, 6394-6396.
Olivieri, G., Bodycote, J., Wolff, S. (1984) Adaptive response of human lymphocytes to low concentrations ofradioactive thymidine, Science, 223, 594-597.
Oudalova, A.A., Geras’kin, S.A., Dikarev, V.G., Nesterov, Ye.B., Dikareva, N.S. (2002) Induction of chromosome
aberrations is non-linear within low dose region and depends on dose rate, Radiat. Prot. Dosim., 99, 245248.
Oudalova, A.A., Geras’kin, S.A., Dikarev, V.G., Evseeva, T.I., Dikareva, N.S. (2005) Cytogenetic effects induced bylow level radiation in plant meristems. 7th Int. Symposium ‘Change and Continuity in Radiation Protection’,
Cardiff, U.K., 356-361.
Pentreath, R.J. (2002) Radiation protection of people and the environment: developing a common approach, J.
Radiol. Prot., 22, 45-56.
Pohl-Ruling, J., Fischer, P., Haas, O., Obe, G., Natarajan, A.T., van Buul, P.P., Buckton, K.E., Bianchi, N.O.,
Larramendy, M., Kucerova, M., Polikova, Z., Leonard, A., Fabri, L., Sharma, T., Binder, W., Mukherjee,
R.N., Mukherjee, U. (1983) Effect of low dose acute X-irradiation on the frequencies of chromosomal
aberrations in human peripheral lymphocytes in vitro, Mutat. Res., 110, 71-82.
Pollycove, M. & Feinendegen, E. (2001a) Biologic responses to low doses of ionizing radiation: detriment versus
hormesis. Part 1. Dose responses of cells and tissues, J Nuclear Medicine 42(7), 17N-27N.
Pollycove, M. & Feinendegen, E. (2001b) Biologic responses to low doses of ionizing radiation: detriment versus
hormesis. Part 2. Dose responses of organisms, J Nuclear Medicine 42(9), 26N-32N.
Sachs, L. (1972) Statistische auswertungsmethoden, Springer-Verlag, Berlin.
Sandhu, S.S., Serres de, F.J., Gopalan, H.N., Grant W.F., Veleminsky, J., Becking, G.C. (1994) Environmental
monitoring for genotoxicity with plant systems: an introduction and study design, Mutat. Res., 310, 169–173.
Sarapul’tzev, B.I., & Geras’kin, S.A. (1993) Genetic Basics of Radioresistancy and Evolution. Energoatomizdat- 191 -

Publish., Moscow (in Russian).
Sax, K. (1938) Chromosome aberrations induced by X-rays, Genetics, 23, 494-516.
Sevankaev, A.V. (1991) Quantitative estimation of the cytogenetic effects of low-level radiation: current aspects,
Radiobiology, 31(4), 600-605.
Shevchenko, V.A., Pechkurenkov, V.L., Abramov, V.I. (1992) Radiation Genetics of Natural Populations: The
Genetic Consequences of the Kyshtym accident. Nauka Publish. Moscow.
Shmakova, N.L., Fadeeva, T.A., Nasonova, E.A., Krasavin, E.A., Rsyanina, A.V. (2002) Cytogenetic effects of low
dose radiation in mammalian cells: the analysis of the phenomenon of hypersensitivity and induced
radioresistance, Radiat.Biol. Radioekol., 42, 245-250.
Sorensen, K.J., Zetterberg, L.A., Nelson, D.O., Grawe, J., Tucher, J.D. (2000) The in vivo dose rate effect of chronic
gamma radiation in mice: translocation and micronucleus analyses, Mutat. Res., 457, 125-136.
Sparrow, A.N., Underbrink, A.G., Rossi, H.H. (1972) Mutations induced in Tradescantia by small doses of X-rays
and neutrons: analysis of dose-response curves, Science, 176, 916-918.
Spitkovskij, D.M., Ermakov, A.V., Gorin, A.I., Pospekhova N.I., Sorokina T.A., Talisina T.A. (1994a) Unscheduledsynthesis of DNA and changes in structural parameters of nuclei in human lymphocytes after low doses ofγ-radiation along and in combination with UV-radiation, Radiat.Biol. Radioekol., 34(1), 23-31.
Spitkovskij, D.M., Zaitzev, S.V., Talysina, T.A. (1994b) Modelling peculiarities of genetic damage initiation by low
doses of ionizing radiation in eukaryotic cells from a concept of evolution reserve cell population existence,
Radiat.Biol. Radioekol., 34(6), 739-747.
Takahashi, E.I., Hirai, M., Tobari, E., Utsugi, T., Nakai, S. (1982) Radiation-induced chromosome aberrations inlymphocytes from man and crab-eating monkey: the dose-response relationships of low doses, Mutat. Res.,94, 115-123.
Timofeeff-Resovsky, N.W. & Luchnik, N.V. (1960) Cytological and biophysical basis of radiostimulation in plants,
Proc. Inst. Biol. Ural Br. Sc. Ac. USSR, 5-17.
Timofeeff-Ressovsky, N.W., Zimmer, K.G., Delbruck, M. (1935) Uber die Natur der Genmutation und derGenstruktur, Nachr. Gess. Wiss. Gottingen, 1, 189-245.
Tubiana, M. (1998) The report of the French Academy of Science: ‘Problems associated with the effects of low
doses of ionising radiation’, J. Radiol. Prot., 18, 243-248.
Upton, A.C. (2003) The state of the art in the 1990’s: NCRP report no. 136 on the scientific bases for linearity in thedose-response relationship for ionizing radiation, Health Physics, 85, 15-22.
Vasiliev, F.P. (1988) Numerical Approaches to Extreme Task Solution, Nauka Publish., Moscow.
Wei, L., Zha, Y., et al. (1990) Epidemiological investigation of radiological effects in high-background areas of
Yangjang, China, J. Radiat. Res., 31, 119−136.
Zaichkina, S.I., Aptikaeva, G.F., Akhmadieva, A.Kh., Smirnova, E.N., Antipov, A.V., Prilutskaia, N.B., Kuglik, V.,
Shlotova, Ia., Ganassi, E.E. (1992) The characteristics of the realization of cytogenetic damage inmammalian and plant cells exposed to low doses of radiation, Radiobiology, 32, 38-41.
Zaka, R., Chenal, C., Misset, M.T. (2002) Study of external low irradiation dose effects on induction of chromosome
aberrations in Pisum sativum root tip meristem, Mutat. Res., 517, 87-99.

- 192 -