brechignac_f_desmet_g_eds_equidosimetry_ecological_standardi

241

4. Discussion

Tradescantia is a member of Commelinaceae family. It has two distinct features as a wonderful experimental object in radiobiological and environmental studies. Based on the heterozygosity for the flower colour of Tradescantia plants (i.e. clone 02 or 4430), somatic cell mutation assay technique has been developed, which is known as Trad-SH assay [12,13]. The other assay technique based on the chromosome aberrations in the meiotic pollen mother cells is called Trad-MCN assay [3,5]. While another higher plants such as Allium cepa, Vicia faba, and Arabidopsis thaliana can also be used as an botanical assay tool for detecting radiations, mutagens and environmental clastogens, Trad-MCN assay has an advantage over anything else because hundreds of thousands of pollen mother cells can be scored easily and quickly and thus statistically reliable results can be obtained.

The results of the present study confirm that the Trad-MCN bioassay can detect clastogenic effects of endocrine disruptors in form of solution and ionizing radiation, as well. Most of works with BPA have focused on animal studies, only because it gathers attention as an endocrine disruptor. The clastogenic effect of BPA in plant systems is newly reported here. Furthermore, the clastogenic effects of BPA as measured by means of the micronucleus frequencies in the pollen mother cells of Tradescantia were compared to those of ionizing radiation in the same cell system for equidosimetry purpose.

The frequencies of micronucleus showed a positive dose-response relationship in the range of 0 to 0.5 Gy, and a clear concentration-response relationship in the experimental range of BPA concentrations. Linear regression doseand concentrationresponse lines for the gamma rays and BPA treatments were established by two parallel series of experiments. These two regression lines were compared at the same levels of MCN frequencies induced by these two agents in order to determine the equi-dosimetric effectiveness of BPA as compared with ionizing radiation. Using the regression equations described in the previous section, it is possible to calculate the equivalent doses to each other. A unit dose of radiation (cGy) induces 1.97 MCN whereas a unit dose of BPA (mM) does 4.26 MCN. In this context, 1 mM of BPA is equivalent to 2.16 cGy of radiation in the induction of clastogenic damage in the pollen mother cells of

Tradescantia 4430.

BPA of 11.8 mM can give rise to 53.3 MCN/100 tetrads, the same frequency that is induced by 25 cGy of gamma rays. It is noteworthy that clinical symptoms can develop after a whole body exposure to ionizing radiation dose higher than 25 cGy in human beings [14].

5. Concluding remarks

This study was designed to demonstrate the biological effects of BPA, one of the well-known endocrine disruptors, by means of the Tradescantia micronucleus assay. The biological effect of gamma rays on the same cell system was also evaluated for comparison with that of BPA to estimate the equidosimetric effectiveness of BPA. Fresh cuttings of Tradescantia BNL 4430 were treated with BPA solutions of 0 to 4 mM for 6 hours for the absorption of the solution through the stem of the plant cuttings. Other groups of the cuttings were irradiated with 0 to 1.0 Gy of gamma rays. The frequencies of micronucleus showed a positive dose-response relationship in the range of 0 to 0.5 Gy, and a

242

clear concentration-response relationship in the experimental range of BPA concentrations. By comparing the two experimental results, it is possible to estimate the BPA concentration and its equivalent radiation dose for a fixed value of MCN frequency. BPA of 11.8 mM can give rise to 53.3 MCN/100 tetrads, the same frequency that is induced by 25 cGy of gamma rays. It is of biological importance that clinical symptoms start to develop after a whole body exposure to radiation higher than 25 cGy. The results indicated that the pollen mother cells were an excellent biological end-point for measuring toxicity of endocrine disrupting chemical bisphenol A. In addition, TradMCN assay can be easily applied to measure the biological effects of suspected endocrine disruptors such as octylphenol and nonylphenol.

6. Acknowledgement

This study has been carried out under the National R&D Program and KoreaRussia Scientists Exchange Program by the Ministry of Science and Technology (MOST) of Korea.

7. References

1.D.C. Shin, 1999. What is the endocrine disruptor? Food Science and Industry, 33 : 2-18 (1999) (in Korean).

2.K. Sax. Chromosome aberration induced by x-rays. Genetics 23 : 494-516 (1938).

3.T. H. Ma, G. J. Kontos and V. A. Anderson. Stage sensitivity and dose response of meiotic pollen mother cells of Tradescantia to x-rays. Environ. Expt. Bot. 20 : 169-174 (1980).

4.J. H. Taylor. The duration of differentiation in excise anthers. Am. J. Bot. 37 : 137-140 (1950).

5.T. H. Ma, A. H. Sparrow, L. A. Schairer and A. F. Nauman. Effect of 1,2-dibromoetane (DBE) on meiotic chromosomes of Tradescantia. Mutat. Res. 58 : 251-258 (1978).

6.T. H. Ma. Micronuclei induced by X-rays and chemical mutagen in meiotic pollen mother cells of Tradescantia - A promising mutation test system. Mutat. Res. 64 : 307-313 (1979).

7.J. A. Heddle. A rapid in vivo test for chromosome damage. Mutat. Res. 18 : 187-190 (1973).

8.J. R. K. Savage and D.G. Papworth. An investigation of LET 'finger-prints' in Tradescantia. Mutat. Res. 422 : 313-322 (1998).

9.W. F. Grant. Higher plant bioassays for the detection of chromosomal aberrations and gene mutations--a brief historical background on their use for screening and monitoring environmental chemicals. Mutat. Res. 426 : 107-112 (1999).

10.T. H. Ma, G. L. Cabrera, R. Chen, B. S. Gill, S. S. Sandhu, A. L. Vandenberg and M. F. Salamone. Tradescantia micronucleus bioassay, Mutat. Res. 310 : 221–230 (1994).

11.J. K. Kim, H. S. Song and S.H. Hyun. Dose-response relationship of micronucleus frequency in pollen mother cells of Tradescantia, J. Kor. Assoc. Radiat. Prot., 24 : 187-192 (1999).

12.A. G. Underbrink and A. H. Sparrow. Power Relations as an Expression of Relative Biological Effectiveness (RBE) in Tradescantia Stamen Hairs, Radiat Res., 46 : 580-587 (1971).

13.T. H. Ma, G. L. Cabrera, A. Cebulska-Wasilewska, R. Chen, F. Loarca, A. L. Vandenberg and M. F. Salamone. Tradescantia Stamen Hair Mutation Bioassay, Mutat. Res., 310 : 211-220 (1994).

14.J. K. Kim and S. H. Hyun. Comparative study on human risk by ionizing radiation and pesticide as biological information about environmental disaster, J. Kor. Assoc. Radiat. Prot., 26 : 385-392 (2001)

Part 6.

Genetic Factors, Environment and Toxicants

CYTOGENETIC EFFECTS IN PLANTS AFTER WEAK AND COMBINED EXPOSURES AND A PROBLEM OF ECOLOGICAL STANDARDIZATION

S.A. GERAS’KIN, V.G. DIKAREV, N.S. DIKAREVA, A.A. OUDALOVA, D.V. VASILIYEV

Russian Institute of Agricultural Radiology and Agroecology, Obninsk, RUSSIA

T.I. EVSEEVA

Biological Institute, Komi Research Center, Urals Department of RAS, Syktyvkar, RUSSIA

1. Difference between sanitary-hygienic and ecological approaches to substantiation of derived levels for ionizing radiation

For the last three decades, a scientific background for radiation protection of biota has been based on principles developed by the ICRP. A substance of the most important among them can be formulated as ‘If protection of a human is provided, it will guarantee protection of an environment’. In such an approach, objects of environment are considered as sources of external and internal irradiation for humans, and radionuclides contents are standardized by the sanitary-hygienic approach. At the same time, although standards derived from these principles guarantee the protection of a human, they are not always able to guarantee the equal protection of other objects of living nature. Standardization with this approach assumes in default that doses absorbed in human are close to doses absorbed in other environmental objects and hence if one provides low doses for people it will automatically assure low doses for other alive organisms. This is not necessarily true. So, dose values per unit of radioactive contamination density obtained by plants and animals from radioactive fallouts can exceed values for humans by a factor of 10-100 [1]. With regard to a close radiosensitivity of human and a number of edificator species determining functioning and stability of ecosystems (coniferous trees, many mammalians), such a relationship between doses absorbed in human and environmental objects makes clear that a special attention is to be paid to protection of plants, animals and communities. Thus, taking the sanitary-hygienic approach as the major one in providing radiation security for the population, one should admit the necessity of its complementation with standards of radiation safety for biota.

A form of existence of plants and animals in nature is a population. Therefore, when one changes from a sanitary-hygienic approach to an ecological one not only indexes of standardization norms modify but also the objects to which they should be applied (Table 1). These objects are not individuals as it used to be in frames of the sanitary-hygienic approach but natural or artificial systems of supra-individual levels, that are populations, ecosystems, and biogeocenoses. A distinction of doses delivered to human and biota mainly reveals at severe radiation accidents [1]. But, experience gained from the elimination of consequences of such accidents showed that, even in this case, there was not an overall lethal lesion at the ecosystematic level except at coniferous forests. This regards to very good

245

F. Brechignac and G. Desmet (eds.), Equidosimetry, 245–255.

© 2005 Springer. Printed in the Netherlands.

246

possibilities of populations for strength restoration. Thus, up to 85% of young generation in populations of small sparrow birds (brood of a current year) die every year, but the whole population remains quite stable. Therefore, differences between doses, which are able to induce an increase of stochastic effect occurrence in man, and those, which can result in a death of plant and animal populations, are so great that the main principle of the ICRP, -‘if a man is protected environment is also saved’, - does not overwhelmingly break in current ecological situations.

Table 1. Differences between the sanitary-hygienic and the ecologic approaches at development of derived levels

On the other hand, the most important principle that a set of ecological criteria ought to meet is the principle of non-admission of deterioration of the ecological state of the environment. The hierarchic structural-functional organization of living matter supposes a multi-level system of responses to external impact. In this connection, a question emerges: alterations at what level of biological organization really indicate a deterioration of ecology?

Natural and agrarian ecological systems have a diverse variability of responses to irradiation. A range of dose rates at which biota irradiation actually takes place covers five values of magnitude. The derived levels of exposure of man and biota recommended by the international agencies are compared with dose rate values of chronic radiation producing effects at different levels of biological organization in Table 2. From these data, genetic effects and alterations of genetic structure in wild plant and animal populations are observed at dose rates, which are below the level considered by the IAEA as not providing any hazard to biosphere. Nevertheless, it is changes at an underlying, molecular-cellular level of living mature organization that give the earliest and confident information about any negative changes in environment. In fact, according to the principle of attenuation of adverse effects with increasing level of organization of biological systems [4], the earliest changes can be detected at the subcellular and cellular levels of organization of living matter. Genetic tests show direct and specific responses to exogenous stresses. Therefore, they may be of a unique importance for estimating the changes that occur, as a rule, before morphological, physiological, populational, or other deviations from the norm. In addition, the genetic tests reveal the most substantial effects of environmental pollution related to increasing

247

mutagenic pressure on the biosphere. This includes an increasing incidence of cancer and hereditary diseases; an increasing genetic load in humans, animals, and plants; and changing genetic and species structure of biocenoses. Therefore, it is the genetic testsystems that must be used for early diagnosis of the alterations resulting from human economic activity.

Table 2. Comparison of the derived levels of exposure of man and biota recommended by the international agencies with dose rates of chronic radiation producing effects at different levels of biological organization [1-3]

Let us consider what changes on cytogenetical level can be induced with low doses of ionizing radiation in plants under conditions of single and combined with factors of different nature of action. Important features of biological effect of low-level radiation that do not follow from well-known effects of high and moderate doses are:

- nonlinearity of dose response;

- synergetic and antagonistic effects of combined action of different nature factors;

-radiation induced replicative instability of genome;

-phenomenon of radioadaptation.

A development of a new concept of radiation protection for human and biota should be based on the clear understanding of these effects and their contribution to response of biological objects to single low-level radiation and combined with other anthropogenic factors influence.

2. Non-linearity of dose response

The analysis of experimentally observed reactions of cells on low level irradiation has shown [5, 6] that regularities of a yield of cytogenetic disturbances in this range are characterized by a intensely expressed non-linearity and have an

248

universal character, differing for miscellaneous objects by values of doses, at which there are changes of the relation character. To check this state an experiment has been carried out on barley seedlings [7]. It is apparent from the results presented in Fig. 1, even without using precise quantitative criteria, that the piecewise linear model based on our concept of biological effect of low-level ionizing radiation [6, 8] describes the experimental data about cytogenetic damage appearance in studied range much better than the linear one. It is important, that the improvement of the quality of approximation is reached not on account of the model complicating but because it is possible to achieve a mutual conformity (functional isomorphism) between a biological phenomenon and its mathematical model on the set of piecewise linear functions. Comparison of approximation quality that can be achieved with models of different complexity by the most common quantitative criteria testifies to it as well (Table 3).

Dose (mGy)

Fig. 1. Frequency of aberrant cells in barley germs exposed to low radiation doses and its approximation with linear (1) and piecewise linear (2) models .

Table 3. Comparison of the approximation quality of the experimental data about the yield of cytogenetic disturbances in irradiated barley seedlings by models of different complexity

R2 – multiple correlation coefficient,

T - criteria of structural identification [22],

249

An absence of authentic experimental information about biological effects that can be induced by irradiation at doses less than 1 cGy, as well as an existence of alternative approaches to explain the causes of a nontrivial shape of dose dependence at low levels, have stipulated a necessity of new experimental researches. In our studies on cells of stamen filament cells of spiderwort (clone 02) [9] it not only appears possible to confirm existence of the plateau revealed on barley seedlings (for spiderwort, due to the greater sensitivity, this range is shifted towards smaller doses), but also to experimentally support a theoretical prediction of our concept about an existence of dose range (in this case, less 1 cGy) where there is no significant increase of genetic disturbances (Fig.2). Let us remark, that the conclusion about the non-linear character of dose dependence which follows from the presented results is based on experimental data only and does not depend on any hypothesis or extrapolation models.

Dose, mGy

Fig. 2. Frequency of somatic mutations in stamen filament cells of Tradescantia (clone 02) versus ϑ-irradiation dose.

3. Synergetic and antagonistic effects of combined action of different nature factors

It was shown in course of our experimental studies carried out on spring barley, onion and spiderwort under combined effect of such common agents as ϑ- radiation, pesticides, heavy natural radionuclides, salts of heavy and alkali metals [9-12] that there is a principal possibility of synergistic and antagonistic effect occurrence. In regard to this consideration, there are two important questions. The first is: are there ranges of doses and concentrations such that nonlinear effects can be observed more often under a combination of exposures from these ranges? It seems there are. It is in the case of combined action of low-level exposures when synergetic effects are induced with an increased probability [9-13]. The second question issues forth from the first: how important is a contribution of nonlinear effects to the response of biological objects? If their contribution is negligible, one can disregard it and estimate a result of a combined influence using available information about single effects of each factor that is more convenient and easier,

250

of course. A study of cytogenetic disturbances induction in intercalar meristem cells of spring barley grown on soil contaminated with low concentrations of Cs-137 and cadmium nitrate [12] has shown that the effect of combined action exceeds the sum of separate effects as much as 70% (Fig.3). On the contrary, the observed effect at soil pollution by radioactive cesium, lead and pesticides averaged only 50% from anticipated one proceeding from the additive model. Therefore, an application of data from a single effect analysis to forecast biological effects of combined effect is unacceptable and causes the essential distortion from picture that can be observed experimentally.

Fig. 3. Cytogenetic damage yield in intercalar meristem of spring barley in conditions of combined soil pollution by Cs-137 and Cd:

4. Radiation induced replicative instability of genome

In 1987-1989 within the ChNPP 10-km zone, we conducted an experiment [14] with one of the purposes being to analyze cytogenetic variability in three successive generations of winter rye and wheat, grown at four plots with different levels of radioactive contamination. A dose on a growing point varied within the limits of 18-717 cGy between plots for the vegetative season 1987-1988, and within the limits of 11-417 cGy for 1988-1989. Fig. 4 shows that in this experiment in autumn of 1989, aberrant cell frequencies in leaf meristem of winter rye and wheat of the second and third generations significantly exceeded these parameters for the first generations; it was significant on experimental plots with the highest levels of radioactive contamination. The distinctions between cytogenetic parameters obtained for the second and third generations are small and statistically not significant,and the observed effect is of a threshold character.

251

Dose, cGy

Fig. 4. Modification of aberrant cell frequency in leaf meristem of winter rye and wheat in three generations grown up on contaminated plots.

It is important that it was not root meristem of seedlings but intercalar meristem of plants where the cytogenetic disturbances were analyzed. It means that the most part of radiation-induced alterations accumulated during the previous vegetative season was realized into real mutations long before the samples were fixed for the cytogenetic analysis. As in autumn of 1989 plants of all three generations developed in identical conditions and were exposed to identical doses, the most probable explanation of the detected phenomenon is related to genome destabilization in plants grown from seeds affected by radiation. From these viewpoints, the results observed in this study, indicating a threshold character of genetic instability induction, may be a reflection of the first stage of cytogenetic adaptation, that is, chronic low-dose irradiation appears to be an ecological factor creating a background for alteration of the genetic structure of a population.

5. Phenomenon of radio-adaptation.

A study of cytogenetic damage in vegetative (needles) and reproductive (seeds) organs in the Pinus sylvestris L. micropopulations growing at sites with contrast radioactive contamination levels in the 30-km ChNPP zone and also in region of arrangement of the ‘Radon’ enterprise for processing and storage of radioactive waste (the town of Sosnovy Bor, Leningrad region) is concerned to a question about a role of such alterations on the population structure. From the results of this study presented in Fig. 5, 6, a conclusion follows that there is an expressed genotoxic influence in the investigated regions. And, while the incidence of cytogenetic damage in the samples from the Chernobyl NPP region correlates