© Borgis - New Medicine 2/2009, s. 43-44
*Ewa Ogłodek1, Danuta Mo?2, Aleksander Araszkiewicz1
SIGNIFICANCE OF AgNOR MEASUREMENT IN RAT THYROID GLAND
1Department of Clinical Psychiatry of the Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Poland
2Individual medical practice
Summary
Aim. The aim of this study was to evaluate the influence of extremely low-frequency magnetic fields with parameters used in the treatment of people in order to alleviate the pain and stimulate metabolism of cells in the activity of nucleoli and nucleolar organizers in female rat thyrocytes.
Material and method. 24 female Wistar rats, Hannover substrain, were included in the experiment. The rats were divided into 2 groups: group ”40”, exposed to extremely low-frequency magnetic fields of frequency 40 Hz; and control group ”C” with sham exposure. After taking the preparations out of water they were stained in a solution of silver nitrate, and again rinsed in water, and after that they were put, one by one, in 5% alkaline solution of sodium thiosulphate.
Results. In the preparations of rat thyroid glands the following parameters were evaluated: the number of nucleoli (NNU), the surface area of nucleoli (SANU) and the number of AgNOR nucleolar organizers (NAgNOR), and the surface area of AgNOR nucleolar organizers (SAAgNOR).
Conclusions. The observed changes of surface area and the number and surface areas of nucleoli and nucleolar organizers point to the increase of the replicative activity in nuclei of thyrocytes and possible increase of DNA synthesis.
INTRODUCTION
Among variable magnetic fields extremely low-frequency magnetic fields (ELF-MF) can be distinguished. Extremely low-frequency magnetic fields are a stress stimulus related to the development of an adaptive mechanism associated with the autonomic nervous system as well as endocrine system. Exposure to the magnetic field may be treated as a specific stress situation, the consequence of which is the increase of membrane metabolism of neurotransmitters. It also emerges from specialist literature that in the response to the stress stimulus which, obviously, is the activity of the extremely low-frequency magnetic fields, changes in biosynthesis of protein and the regulation of the cell cycle and stimulation of transcription in the cell occur (10).
AIM
The aim of this study was to evaluate the influence of extremely low-frequency magnetic fields with the parameters 40 Hz frequency, induction of 10 mT and sinusoidal waveform impulse on the activity of nucleoli and nucleolar organizers in female rat thyrocytes.
MATERIAL AND METHOD
24 female Wistar rats, Hannover substrain, were included in the experiment. The rats were divided into 2 groups: group ”40”, exposed to extremely low-frequency magnetic fields of frequency 40 Hz; and control group ”C” with sham exposure. After taking the preparations out of water they were stained in a solution of silver nitrate, and again rinsed in water, and after that they were put, one by one, in 5% alkaline solution of sodium thiosulphate. After having been rinsed in water the preparations were stained in a solution of haematoxylin and eosin. The microscopic slides were assessed using 400x magnification of the light microscope, maintaining the calibration scale before each evaluation of a slide. For measurements pore size mesh was used, with pores of 500 μm, and the size of the mesh was 15x15 mm. Fifty visual fields in each of the examined rats and for each parameter were evaluated and in each visual field 20 cells were assessed. Surface areas of nuclei, nucleoli and nucleolar organizers were calculated by means of the direct measurement of 2-dimensional area occupied by an object in the microscope field of vision. The areas were calculated moving the slide clockwise.
The following methods were used for the purpose of the statistical analysis: ANOVA for a single variable was performed using the following tests: Cochran-Cox test and investigation of significance of differences between the mean values (post-hoc analysis) by means of Tukey´s test.
RESULTS
In the preparations of rat thyroid glands the following parameters were evaluated: the number of nucleoli (NNU), the surface area of nucleoli (SANU) and the number of AgNOR nucleolar organizers (NAgNOR), and the surface area of AgNOR nucleolar organizers (SAAgNOR). The number of nucleoli and the number of nucleolar organizers (AgNOR) in main cells of the thyroid gland increased significantly under the activity of ELF-MF. Significant increase of nucleoli surface field and AgNOR nucleolar organizers field under the activity of ELF-MF was observed (tab. 1).
Table 1. Examination parameters: the number of nucleoli (NNU), surface area of nucleoli (SANU), the number of nucleolar organizers (NAgNOR), the surface area of AgNOR nucleolar organizers (SAAgNOR).
| Parameters | Examined groups of rats X +/- SD | Examined groups of rats X +/- SD | Level of significance | Cochran-Cox Test | Tukey Test |
| ´40´ | ´C´ | ´40´ and ´C´ | ´40´ and ´C´ | ´40´ and ´C´ |
| SANU [ľm2] | 0.41 (+/-0.07) | 0.17 (+/-0.07) | p<0.0001 | 0.555 | 0.00002 |
| NNU | 5.17 (+/-0.08) | 3.27 (+/-0.09) | p<0.0001 | 0.363 | 0.00002 |
| NAgNOR | 3.72 (+/-0.08) | 1.46 (+/-0.07) | p<0.0001 | 0.422 | 0.00002 |
| SAAgNOR [ľm2] | 0.19 (+/-0.06) | 0.12 (+/-0.05) | p<0.0001 | 0.471 | 0.00002 |
DISCUSSION
In the present investigation, extremely low-frequency magnetic fields influenced the increase of the number and surface area of nucleoli and nucleolar organizing regions (AgNOR).
Shechtman L. et al. (8) and Camargo R.S. et al. (3) showed that extremely low-frequency magnetic fields influence the increase of the number of nucleoli and nucleolar organizing regions (AgNOR). These authors stated that AgNOR number increases during the G1 phase of the cellular cycle and remains up to the G2 phase. Simultaneously the AgNOR surface area gets higher.
Rajkovic V. et al. (5, 6, 7) and Izmest´eva O.S. et al. (4) stated that AgNOR number increases during the G1 phase of the cellular cycle and remains up to the G2 phase. The presentation of the structure of chromatin fibres in AgNOR and the presence of proteins stained by silver nitrate AgNOR during the cellular division is the expression of the readiness of rDNA genes for transcription which will take place after the end of mitosis in the next interphase. At the end of prophase when the AgNOR action finishes the material is partially transported to the cytoplasm. In metaphase and anaphase in the place of AgNOR remain only non-numerous silver spots pointing to the relationship with the transcription process during the interphase preceding the division. Only in the late telophase, together with the activation of AgNOR, does the quantity of the silver absorptive material increase once again and the nucleoli appear (1, 9).
CONCLUSIONS
1. The observed changes of surface area and the number of nucleoli and nucleolar organizers point to the increase of the replicative activity in nuclei of thyrocytes and possible increase of DNA synthesis.
2. Increase of replicative activity in nuclei of thyrocytes – increased number and surface area of nuclei, nucleoli and AgNOR in computer morphometric analysis – may be useful in the diagnostics of human thyroid disorders.
3. AgNOR expression is a prognostic marker in human thyroid tumour. Thyroid cancer has demonstrated a positive correlation between AgNOR and other markers of cellular proliferation.
4. AgNOR determinations have been considered to provide an objective prognostic parameter to predict the clinical behaviour of surgically resected thyroid tumour in individual patients.
Piśmiennictwo
1. Augustynowicz A et al.: Assessment of proliferative activity of thyroid Hürthle cell tumors using PCNA, Ki-67 and AgNOR methods. Folia Histochem Cytobiol. 2004; 42(3): 165-8. 2. Belyavskaya NA: Biological effects due to weak magnetic field on plants. Adv Space Res. 2004; 34(7): 1566-74. 3. Camargo RS et al.: Significance of AgNOR measurement in thyroid lesions. Anal Quant Cytol Histol. 2006; 28(4): 188-92. 4. Izmest´eva OS et al.: Effects of electromagnetic field of thermal intensity on the hypophysis-thyroid unit of the neuroendocrine system. Radiats Biol Radioecol. 2003; 43(5): 597-600. 5. Rajkovic V et al.: Histological characteristics of cutaneous and thyroid mast cell populations in male rats exposed to power-frequency electromagnetic fields. Int J Radiat Biol. 2005; 81(7): 491-9. 6. Rajkovic V et al.: Stereological analysis of thyroid mast cells in rats after exposure to extremely low frequency electromagnetic field and the following „off” field period. Acta Biol Hung. 2005; 56(1-2): 43-51. 7. Rajkovic V et al.: The effect of extremely low-frequency electromagnetic fields on skin and thyroid amine- and peptide-containing cells in rats: an immunohistochemical and morphometrical study. Environ Res. 2005; 99(3): 369-77. 8. Shechtman L et al.: Diagnostic value of AgNOR staining in thyroid cytology. Anal Quant Cytol Histol. 1998; 20(3): 187-91. 9. Solymosi T et al.: Diagnostic value of AgNOR method in thyroid cytopathology: correlation with morphometric measurements. Diagn Cytopathol. 1996; 14(2): 140-4. 10. Tenuzzo B et al.: Biological effects of 6 mT static magnetic fields: a comparative study in different cell types. Bioelectromagnetics. 2006; 27(7): 560.
