GENOTOXIC EFFECTS OF ELECTROMAGNETIC EXPOSURE TO ELF FIELDS INVESTIGATED AT THE LEVEL OF MERISTEMATIC TISSUES *

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1 GENOTOXIC EFFECTS OF ELECTROMAGNETIC EXPOSURE TO ELF FIELDS INVESTIGATED AT THE LEVEL OF MERISTEMATIC TISSUES * C. IONITA-MIRONESCU 1, D. VRINCIANU 1, I. BARA 2, D. CREANGA 1, M. RACUCIU 3 1 Univ. Al. I. Cuza, Faculty of Physics, 11 Blvd. Carol I, Iasi, Romania, cristina_elena_ionita@yahoo.com, dorina.creanga@gmail.com 2 Univ. Al. I. Cuza, Faculty of Biology, Iasi, Romania, iuliabara@uaic.ro 3 Univ. L. Blaga, Faculty of Sciences, Sibiu, Romania, mracuciu@yahoo.com Received September 5, 2011 Cytogenetic investigation was carried out focused on the response of wheat and barley seedlings to the exposures to 50 Hz/3.65 mt and respectively 50 Hz/7.0 mt magnetic fields. The presence of abnormal ana-telophases was noticed in all samples with significant increased incidence for the higher magnetic field. The differentiated behavior of wheat and barley was discussed both for mitotic index and chromosomal aberration percentage. Key words: ELF fields, chromosomal aberrations, mitotic index. 1. INTRODUCTION The scientific interest in the effect of electric fields on living bodies is mainly related to the numerous natural and artificial sources, including nearby high voltage transmission lines, primary and secondary over head utility distribution lines and the electrical grounding system [1-3]. Nowadays, there is a wide variety of electronic devices that generate electromagnetic fields (EMF) of very low frequencies such as 50 and 60 Hz (extremely low frequencies fields, ELF), such as household appliances as televisions, electric blankets, hair driers, and therapeutic medical applications [4, 5]. The widespread use of electric power and higher levels of EMFs encountered in the environment have raised concerns that such exposures might be associated with cancer risks [6, 7]. Some articles reported the influence of ELF fields where the inhibition of root development was evidenced following the exposure to 60 Hz magnetic field; so, the changes of mitotic activity induced by 60 Hz, 6 KV/m ELF field in wheat seedlings was evidenced by Hanafy et al. [1] that mentioned the decrease of cells in anaphase but the increase of cells in metaphase and ana- * Paper presented at the 12 th International Balkan Workshop on Applied Physics, July 6 8, 2011, Constanta, Romania. Rom. Journ. Phys., Vol. 57, Nos. 7 8, P , Bucharest, 2012

2 1178 C. Ionita-Mironescu et al. 2 telophase. This result was correlated with the diminution of DNA synthesis as well as with the decrease of protein synthesis in the electromagnetically exposed vegetal samples. Similar results were obtained by Shebab et al., [8]; the chromosomal aberrations revealed in the frame of that study were mainly interchromatidian bridges in anaphase, retard chromosomes in metaphase and anaphase as well as micronuclei in ana-telophase. In the next we discuss the results of our experimental work focused on the evidence of ELF magnetic field genotoxic effect in two cereal species. 2. MATERIALS AND METHODS 2.1. SAMPLE EXPOSURE SYSTEM Exposure of freshly germinated seeds (30 seeds in each exposure sample) to ELF magnetic field was performed using Helmholtz coil system supplied to 50 Hz/220V power source. The magnetic field induction was measured with field strength analyzer NARDA model EFA-300 based on calibration curve (current, magnetic induction). For current intensity of 0.75 A and 1.5 A, the coil system is able to generate about 3.5 mt and respectively 7.0 mt magnetic field induction in central area where the samples were placed for exposure. The samples were exposed for 30 minutes in each case BIOLOGICAL MATERIAL Caryopses of wheat (common variety) and barley (Madalin and Regal varieties), were provided by experimental lots of Biology Faculty of Al. I. Cuza University of Iasi. After soaking for four hours the caryopses were let to germinate on watered paper support in glass dishes, in darkness at 20.0±0.5 C, into an INCUCELL thermostatic room. After germination, ELF-MF exposure was carried out and the next day root tissue aliquots were taken and treated by applying the rapid Feulgen staining method [9] being further pressed on microscope slides (squash method) to obtain single layer cells CYTOGENETIC TESTS OLYMPUS BX51 microscope with 400x magnitude was used for qualitative and quantitative cytogenetic analysis, based on counting cells frozen in various stages of mitotic division. Mitosis rate (in %) was assessed as: (number of dividing cells)/(total number of analyzed cells) while the frequency of chromosomal aberrations (in %) was given by: (number of cells presenting aberrant divisions)/ (total number of analyzed cells).

3 3 Genotoxic effects of electromagnetic exposure to ELF fields investigated STATISTIC ANALYSIS For each experimental variant five microscope slides with ten observation fields were considered. Average values and standard deviations (calculated with MsExcell soft) were used for graphical plots. Student t-test was applied to assess the statistical significance of the changes in the exposed samples compared to control ones, related to the significance threshold of RESULTS AND DISCUSSION In Figures 1 4 some images presenting cell division state are given. Shortly, in prophase the nucleus is still a round structure and chromosomes are not distinctly separated; in metaphase the nuclear matter is aligned along the division axis, chromosomes being distinctly observable; in anaphase the two identical arrays of chromosomes are slightly separated in order to form the two new nuclei of the future daughter cells; in telophase the separation is totally accomplished. Fig. 1 Normal mitotic stages: 1 prophase; 2 metaphase; 3 anaphase; 4 telophase. Fig. 2 Telophase with inter-chromatidian bridge.

4 1180 C. Ionita-Mironescu et al. 4 Fig. 3 Telophase with retard chromosome. Fig. 4 - Anaphase with expulsed chromosome. Following the careful observation of more than 50,000 cells in every experimental variant resulted in the numerical classification of both normal and aberrant dividing cells. The interchromatidian bridges as that evidenced in Figure 2 represent chromosomal aberrations consisting in the non separation of the two chromosome arrays between the daughter cell nuclei so that diploid cell resulted with possible modified biological features. Retard and expulsed chromosomes either known as lagged and respectively expelled chromosomes as those presented in Figures 3 4 resulted by aberrant cell divisions during which part of genetic information stocked on those lost chromosomes or chromosome fragments is never recovered by the resulted daughter cells. In Figure 5 the summary of the dividing versus interphase cells is presented in the form of the mitosis rate or the index of mitosis, defined as: (number of dividing cells)/(total number of analyzed cells). In both barley varieties (Figure 5) the I.M. was diminished in the samples corresponding to 3.65 mt magnetic induction but it was increased for 7.00 mt magnetic field (statistic significance according to p<0.05). Wheat samples responded differently with non-significant increase for 3.65 mt but with significant diminution for 7.00 mt exposure so that the plant species peculiarities

5 5 Genotoxic effects of electromagnetic exposure to ELF fields investigated 1181 are supposed to underline these results (different species may give different responses to the same external constraint) I.M. percentage (%) control 3.65 mt 7 mt 0 Mădălin barley Regal barley Wheat Fig. 5 The index of mitosis (I.M.%) in the ELF magnetic field exposed samples versus control, non-exposed ones. 25 Chromosomal frequency observed in Ana-Telophase percentage (%) control 3.65 mt 7 mt 0 Mădălin barley Regal barley Wheat Fig. 6 Chromosomal aberrations occurrence index (%), indicates the percentage of chromosomal aberrations induced by electromagnetic exposure.

6 1182 C. Ionita-Mironescu et al. 6 In Figure 6 the total percentage of aberrant divisions mainly ana-telophases is presented. In both cereal species the magnetic ELF field induced significant increased number of chromosomal aberrations compared to the control where also there were noticed some levels of aberrant ana-telophases due to the some variations of environmental factors that could not be monitored. The most affected was the wheat (three times increased frequency of chromosomal aberrations compared to the control) while in the barley varieties slighter genotoxic effects were recorded more than twice higher aberration frequency for Madalin barley and only times increase in Regal barley. Compared to the ELF magnetic field effects on the mitosis rate (Figure 5) where both stimulatory and inhibitory influences were evidenced, the genotoxic effects revealed in Figure 6 are all over increasing for the electromagnetic exposed samples. It is still difficult to explain the differences between the effects of 3.65 mt and 7.00 mt magnetic fields but one possibility is that related also to the superposition of the effects on uncontrollable environmental factor gradients. Unlike the case of ionizing radiation the absorption of ELF magnetic field energy within the exposed tissues can not directly break the covalent bonds of the DNA molecules due to the much higher order of magnitude of that chemical energy. However it is supposed that, following complex synergic phenomena of both biophysical and biochemical nature, the electromagnetic energy absorption is able to trigger a cascade of transformations that finally could result in chemical bonds breaking down as well as in the net influences on the biosynthesis reactions. 4. CONCLUSION The effects of ELF magnetic field exposure in wheat and barley were evidenced by cytogenetic investigation. The plant species seems to influence the responses to electromagnetic exposure as revealed by both the index of mitosis and the frequency of chromosomal aberrations. The changes in the mitosis rate were slighter than those recorded in the number of aberrant divisions suggesting the dominancy of the genotoxic effect on the physiological one. Acknowledgement. "This work was supported by the the European Social Fund in Romania, under the responsibility of the Managing Authority for the Sectoral Operational Programme for Human Resources Development [grant POSDRU/CPP 107/DMI 1.5/S/78342]". REFERENCES 1. M. S. Hanafy, H. A. Mohamed, A. Elham, A. Abd El-Hady, First Scientific Environmental Conference, Zagazig University (2006). 2. M. S. Davies, Bioelectromagnetics. 17 (2), (1996). 3. M. Racuciu, Rom. J. Biophys. 21 (1) (2011).

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