Effects of long-term 50 Hz magnetic field exposure on the micro nucleated polychromatic erythrocyte and blood lymphocyte frequency and argyrophilic nucleolar organizer regions in lymphocytes of mice.

OBJECTIVES: We aimed to investigate the effects of weak extremely low frequency electromagnetic fields (ELF-EMFs) on the nucleus size, the silver staining nucleolar organizer regions (AgNORs), the frequency of micro nucleated peripheral blood lymphocytes (MPBLs) and the micro nucleated polychromatic erythrocytes (MPCEs). METHODS: One hundred and twenty Swiss albino mice were equally divided into 6 groups. The study groups were exposed to 1, 2, 3, 4 and 5 microT 50 Hz-EMFs for 40 days. Micronucleus number (MN) per PBL was determined.. RESULTS: ELF-EMF exposure caused a nonlinear decline of nucleus area. A sharp drop occurred in AgNOR area of 1 microT group, and following it gained an insignificantly higher level than that of the control group. The field did not change mean AgNOR numbers per nucleus of the groups. Relative AgNOR area had the highest level in 1 microT-exposure group, and the level was quite similar to that of the 5 microT-exposure group. The remaining groups had significantly lower values quite similar to that of the control level. The field exposure at any intensity did not affect significantly the frequency of either MPBLs or MPCEs. The number of MN per PBL in the 4 and 5 microT-exposure groups were significantly higher than those of the lower intensity exposure groups. The males in 4 microT-exposure group displayed the highest MN number per PBL, whereas values changed in a nonlinear manner. CONCLUSIONS: The results of the present study suggest that

[Bystander effect development in human mesenchymal stem cells after exposure to adaptive dose of X-radiation].

Transposition and mutual approaching of pericentromeric loci 1q12 of homological chromosomes from the nuclear membrane towards the nuclear centre as well as activation of the chromosomal nucleolus-forming regions (NFR) are observed in human mesenchymal stem cells (hMSCs) as an initial stages of the adaptive response (AR) after exposure to low doses of X-radiation (10 cGy). All these reactions are also induced after addition of cultivation medium from irradiated cells to intact bystander-cells and this phenomenon called bystander effect (BE). Recently the same AR and BE induction results were obtained for human G0-lymphocytes. All these data indicate the existence of universal reaction of homological chromosome loci transposition which was revealed during AR development in differentiated (lymphocytes) and non-differentiated (hMSCs) and also it shows possibility of radiational BE development in suspension and monolayer cell cultures upon addition of stress-signalization factors in incubation medium. We suppose that these factors are extracellular genome DNA fragments apoptotic cells.

[The response of human cancer stem cells on low-dose X-ray exposure].

Recently we found that transposition of homologous chromosomes 1q12 loci towards the nuclear centre and activation of the chromosomal nucleolus-forming regions (NFR) are observed in human lymphocytes after exposure to low doses of X-radiation (10 cGy). These cell reactions were studied for human breast cancer stem cell cultures. There are two cell types in cell culture from single donor: with two (type 1) and three (type 2) loci of 1q12. It was shown that an adaptive response induced by X-ray irradiation is developed only in cells of the type 1 but not in type 2 ones after 3 and 10 cGy doses. We observed a considerable death of cell type 2 after low-dose exposure. Activation of the NFR in breast cancer stem cells after irradiation was not found. In this paper we discuss features of studied cancer stem cells lines and their responses to low doses of ionizing radiation.

In vivo response to the different LET regions of a 55 MeV helium ion beam.

We evaluated the in vivo response to heavy particle irradiation in rat tail epidermis using silver-stained nucleolar organizer regions (AgNOR) as the end-point. The energy degradation of the beam across the circular section of the tail allowed us to study the damage elicited by two different LET regions of a helium beam, i.e. non-Bragg peak (NBP) and Bragg peak (BP), at different sites on the same sample. The tails were locally irradiated with a helium ion beam at different fluences. AgNOR exhibited marked variations between tissue areas only a few micrometers apart within each tail exposed to a given beam fluence. An analysis of the AgNOR variations in NBP and BP areas of tails exposed to different beam fluences revealed a dose-dependent effect. The AgNOR provide quantitative evidence of differential damage in neighboring tissue areas exposed to different LET regions of a helium-ion beam.

AgNOR are sensitive markers of radiation lesions in squamous epithelia.

The possibility of detection of incipient cellular alterations is central to early diagnosis and to clinician's capacity to discriminate between samples that appear similar on routine preparations. We examined the value of silver-stained nucleolar organizer regions (AgNOR) in detecting radio-induced alterations in a model of squamous epithelium biologically similar to oral mucosa. Morphometry of AgNOR has been proven to be of value in the detection of incipient cellular alterations. This method allows for the quantitative evaluation of lesions induced by high doses of radiation long before they become apparent in routine preparations. We herein examine the capacity of AgNOR to reveal the response to low doses of radiation, closer to the therapeutic or accidental dose to which the epithelium of oral mucosa may be exposed.

The relationship between the effects of UV light and thermal shock on gametes and the viability of early developmental stages in a marine teleost fish, the sea bass (Dicentrarchus labrax L.).

To improve the efficiency of gynogenetic induction, the effects of UV light and thermal shock on gametes were investigated in the sea bass. Exposure of sperm to UV light (>/=15 000 erg mm-2) reduced the amount of motile spermatozoa, without affecting the duration of motility in the spermatozoa that remained motile. The Hertwig effect was elicited in eggs fertilized with sperm exposed to >/=35 000 erg mm-2 of UV light, indicating the inactivation of the DNA of the spermatozoa while retaining their ability to trigger development. Resulting embryos (24 chromosomes; one NOR) exhibited the haploid syndrome and died at hatch. Diploidy was restored in eggs fertilized with irradiated sperm by blocking meiosis II with a thermal shock (0 +/- 0.5 degrees C for 10 min, starting 5 min after fertilization). Resulting larvae ( approximately 35% survival at hatching) had 48 chromosomes, one or two NOR and no paternal chromosome fragments (gynogenetic diploids). In eggs fertilized with sperm not exposed to UV light, the same thermal shock induced 100% triploidy (72 chromosomes; one, two or three NOR), with 70% survival at hatching. Multifactorial ANOVA showed that, compared to external factors (sperm diluent, UV light and thermal shock), the contribution of broodstock to the viability of the early developmental stages was not significant (P > 0.05). Effects of the thermal shock were most evident after fertilization (30.7%) but disappeared (0%) at hatching, suggesting that the lower survival of triploids is a consequence of handling, not of the triploid condition per se. However, effects of UV light increased through development (42.5-69.7%), probably reflecting cumulative deficits in protein synthesis.

Argyrophilic nucleolar organizer region counts in squamous cell carcinomas of the head and neck after irradiation and chemotherapy.

After irradiation or chemotherapy of tumors of the head and neck, histological alterations might be difficult to assess. In 30 cases of squamous cell carcinomas of the pharynx, changes in argyrophilic nucleolar organizer region (AgNOR) counts were investigated after therapy. After treatment a significant decrease of AgNOR counts was detected (P < 0.001). No significant differences could be found between groups treated with radiotherapy, chemotherapy or a combination of radiotherapy and chemotherapy. These findings suggest that AgNOR counts might be useful to assess cytotoxic effects on a subcellular level.

Retinal light damage vs. normal aging of rats: altered morphology, intermediate filament expression, and nuclear organization of Müller (glial) cells.

In retinal light damage, degeneration of photoreceptors results in alterations of glial (Müller) cells. In particular, Müller cells show signs of gliosis such as thickening of their stem processes, and expression of glial fibrillary acidic protein (GFAP) which is normally not detectable by immunocytochemistry. We were interested in a quantification of these morphological alterations, and in possible effects of an application of free radical scavengers (Ginkgobiloba extract EGb 761). For this purpose, we studied Müller cells in retinae of albino rats exposed to enhanced illumination for 24 months, a procedure which causes a complete loss of photoreceptor cells. The cells were labeled by (i) bulk filling with the fluorescent dye, Procion yellow, and by (ii) immunocytochemical demonstration of vimentin and GFAP. One group of rats was fed daily with EGb 761 during the last 8 months of life when the remaining photoreceptors (about 50%) died. The retinae were compared with retinae from 3 months-old albino rats, serving as normal young controls, and with retinae from 24 month-old pigment rats, representing normal aging processes. As age-related changes of the ultrastructure of glial cell (astrocytic) nuclei have been described in the literature, the organization of Müller cell nuclei was also studied by an argyrophilic stain, and by electron microscopy. We found that in the thin light-damaged retinae, Müller cells were shorter but thicker than in age-matched control retinae. The volumes of their vitread stem processes were almost unchanged. Müller cells were GFAP-immunoreactive in the light-damaged retinae but not in the controls. The application of EGb 761 prevented the expression by Müller cells of (detectable levels of) GFAP. By contrast, in retinae from EGb 761-treated animals the volumes of the vitread stem processes were significantly increased in comparison to untreated animals. The number of nuclear organization regions was significantly enhanced in Müller cell nuclei from light-damaged untreated albino rats, as compared with the young controls. Application of EGb 761 prevented much of this increase. Thus, exogeneous free radical scavengers do not prevent the occurrence of an reactive hypertrophy but inhibit the expression of "pathological marker molecules", and the (accompanying) signs of enhanced nuclear activity.

Effects of preoperative radiation and chemotherapy on AgNOR counts in oral squamous cell carcinoma.

PURPOSE: This study evaluated the relationship between argyophilic nucleolar organizer regions (AgNORs) and the histologic effects of preoperative chemotherapy or external radiation on oral squamous cell carcinoma. PATIENTS AND METHODS: Thirty-three cases of oral squamous cell carcinoma that were treated with chemotherapy (pepleomycin or 5-FU) or 60Co external radiation were studied. Biopsies were done on the tumor sites before therapy and the number of AgNORs per nucleus was recorded. After therapy, the tumors were resected and the therapeutic effects were assessed histologically. RESULTS: The number of AgNORs per nucleus before therapy ranged from 4.7 to 12.45 (mean +/- SD, 8.71 +/- 2.26). As the number of AgNORs per nucleus increased, the histological effects of preoperative therapy were enhanced. It was 5.75 +/- 0.77 in cases with a poor histological effect of preoperative treatment, 8.03 +/- 1.85 in cases with a mild histological effect, 9.09 +/- 1.85 in the moderate histological effect group, and 10.46 +/- 1.56 in the excellent histological effect group. CONCLUSION: These results suggest that AgNORs could be used to predict the effects of preoperative radiation and chemotherapy on oral squamous cell carcinoma.

Prognostic significance of argyrophilic nucleolar organizer regions (AGNOR) in oesophageal cancer.

AgNOR (Argyrophilic nucleolar organiser region) has been shown in recent times, to have value in knowing the prognosis of carcinoma oesophagus. We have evaluated the significance of AgNOR in oesophageal cancer with reference to prognosis following treatment. Fifty patients of histologically proven squamous cell carcinoma of the oesophagus were studied. Following oesophagectomy the specimens removed were evaluated for AgNOR number. Of the resected specimens, 25 (50%) had an AgNOR count < or = 3.0, 18 (36%) had an AgNOR count of more than 3 per nucleus and in the remaining 7 cases, AgNOR number was not quantifiable due to the total absence of tumour tissue in the postoperative specimen due to preoperative radiotherapy. When followed up for an average period of 25 months (3 to 47 months), it was seen that patients with AgNOR count of < or = 3.0 per nucleous had a similar mean survival (30.39 +/- 3.29 months) as those with counts > 3.0 per nucleus (27.80 + 3.33 months). The survival in the seven patients in whom no tumour was present following preoperative radiotherapy, was 30.30 +/- 2.42 months. An analysis was done for the presence of change in the AgNOR count before and after radiotherapy in twenty eight case of carcinoma oesophagus treated with preoperative radiotherapy. It was found that the counts on an average were lower in patients after radiotherapy (2.89 +/- 1.04 per nucleus), than before radiotherapy (3.17 +/- 9.69). This was found in 24 cases, while the remaining 4 cases showed no change in count after radiotherapy. This suggested that radiotherapy caused a reduction in AgNOR counts. Mean survival in those with decreased count after radiotherapy was 33.65 (+/- 3.35) months. Since the AgNOR counting is a simple method and can be applied to paraffin embedded section, estimation of the AgNOR number may help in determination of prognosis in patients with oesophageal carcinoma. Preoperative radiotherapy seems to decrease AgNOR count with improved survival. These observations need however, to be reproduced with a larger sample size.

Cell proliferation in lung fibrosis-associated hyperplastic lesions.

1. Cell proliferative activity of atypical bronchioalveolar epithelia in lung fibrosis cases treated with bleomycin (BLM) or radiation was investigated by studying the histochemistry of the argyrophil nucleolar organiser regions (AgNORs) and proliferating cell nuclear antigen (PCNA). 2. Five and 14 autopsy cases of individuals who died of pulmonary fibrosis, caused by BLM treatment and irradiation respectively, were compared with (i) six control subjects who proved to have no apparent fibrosis of the lung at autopsy and (ii) four lung squamous cell carcinoma cases. 3. Histopathologically, both the BLM-treated and irradiated cases showed extensive collapse of the lung caused by severe fibrosis, although proliferative epithelial lesions such as atypical bronchioloalveolar hyperplasia and squamous metaplasia were more prominent in the former. 4. The mean AgNOR numbers in both atypical hyperplasias and metaplasias, of either BLM or irradiation cases, were significantly higher than in control bronchioalveolar epithelial areas, whereas they were lower than in the lung cancers. Data for PCNA-labelling indices were in time with those for AgNORs. 5. The results indicate that atypical hyperplastic lesions in the bronchioloalveoli arising during the fibrosing process as induced by BLM, and by irradiation, are highly proliferative.

Argyrophilic nucleolar organizer regions. A revised version of the Ag-NOR-staining technique.

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique.

The well-known technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects, our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue in the microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

Nucleolar organizer regions as markers of incipient cellular alterations in squamous epithelium.

Transcriptionally active nucleolar organizer regions identified by silver staining (AgNOR) vary in number with cellular activity and/or malignant transformation and have been used as a diagnostic tool. A morphometric study of AgNORs was performed in an experimental model of irradiated squamous epithelium (Wistar rat sole skin) 4, 8, and 14 hours and 1, 2, 5, and 7 days post-irradiation with 50 Gy of x-rays. A statistically significant and progressive rise in AgNOR average volume of up to 238% and reduction in AgNOR number/nucleus of up to 40% were detected as a function of post-irradiation time. A statistically significant 46% increase in AgNOR volume was detected as early as 8 h post-irradiation, when no histological changes were observable in routine preparations. These results suggest that AgNORs may be useful as a quantitative marker of incipient changes in cellular activity and caution against the indiscriminate use of AgNORs in the follow-up of lesions which may have been exposed to radiotherapy. Furthermore, this study suggests the possibility of using AgNORs as a sensitive biological dosimeter in cases of uncontrolled exposure to radiation.

Study on mechanism of micronucleoli formation by laser microirradiation.

In the male rat kangaroo cell line PTK2, argon laser (514.5 nm) microirradiation of both nucleoli in interphase cells 30, 23, and 12 h before mitosis, and nucleoli in early prophase cells resulted in the formation of micronucleoli, i.e., several small nucleolus-like bodies, in daughter cells. The irradiated cells were stained with methylene blue, which indicated that the nucleolar RNA was destroyed by laser microirradiation. Feulgen staining was applied to the irradiated cells in combination with the measurements of an MPV-II model microphotometer. Irradiated nucleoli were negative for DNA-Feulgen stain, which indicated that nucleolar DNA was destroyed by laser irradiation, so the nucleolar organizer gene was destroyed. After the nucleoli had been irradiated, the cells were continuously incubated at 37 degrees C for 12 and/or 24 h, then fixed and stained with AgNO3. Most of the nucleoli irradiated silver-stained negative that demonstrated that when the nucleoli were irradiated, rDNA was destroyed and transcription stopped. However, some silver grains were found in the nucleoplasm, whereas the nucleoli were silver-stained negative. The results suggest that subsidiary nucleolar organizer loci might exist scattered throughout the genome.

[Silver staining of the nucleoli of pig embryonic kidney cells during the hyperactivation and inhibition of the synthesis of nucleolar RNA by UV microirradiation]. / Okrashivanie serebrom iadryshek kletok SPEV pri giperaktivatsii i ingibirovanii sinteza iadryshkovoi RNK u UF-mikrooblucheniem.

Silver staining of the nucleoli in pig embryo kidney cells (PK) was studied during the cell cycle and also upon mature nucleoli modifications induced by UV microirradiation. During anaphase only four silver-stained granules were revealed in each daughter set of chromosomes in the four nucleolus-organizing regions (NORs). In the following 1-2 hours, the number of granules in the NORs rapidly increased up to 25-30 per nucleus. During the next 20-25 hours of the cell cycle, the number of silver-stained granules was slowly doubling as the nucleoli grew in size. UV microirradiation of one nucleolus in the nucleus with two nucleoli induced a profound degradation of the injured nucleolus and a compensatory hypertrophy of the intact one. Such nucleolar modifications were accompanied by redistribution of the silver-stained granules between the injured and non-injured nucleoli and by alterations in the levels of nucleolar RNA synthesis in the NORs. These data support a hypothesis that silver-stained proteins may be involved in the regulation of the nucleolar activity.

Establishment of nucleolar deficient sublines of PtK2 (Potorous tridactylis) by ultraviolet laser microirradiation.

One of the two nucleoli of tetraploid PtK2 WA cells in early prophase was irradiated with an ultraviolet (UV) laser microbeam. The daughter cells that maintained the nucleolar deficiency were isolated and cloned. Five nucleolar deficient sublines of PtK2 WA were established, thus providing an experimental system to study the ribosomal gene-nucleolar organizer complex.

[Selectivity and localized nature of exposure in laser microirradiation of cells]. / O selektivnosti i lokal'nosti vozdeistviia pri lazernom mikroobluchenii kletok.

Some modern applications of laser microirradiation to studies of relationship between structure and function are reported. The main advantage of the method is the ability to act on various subcellular parts and organelles, both locally and selectively. However, the degrees of laser microirradiation locality and selectivity are not always sufficiently high. These properties of laser microirradiation are analysed in addition to factors of deteriorating and improving the methods. It is assumed that the most important influence in cellular response to microirradiation is exerted by laser on different cellular membrane systems, particularly on the surface membrane and mitochondria.

The study of X-rays and TCDD effects on satellite associations may suggest a simple model for application in environmental mutagenesis.

Satellite associations were used as parameters to test nucleolar organizer activity. Assuming that toxic and/or mutagenic agents may affect the ribosomal genes, satellite associations in human lymphocytes were analysed following exposure to X-rays and compared with the satellite association pattern of subjects exposed to TCDD. A significant decrease in the satellite association frequency in D group chromosomes was found both in irradiated lymphocytes and in subjects exposed to Dioxin. The findings seem to be in accordance with the hypothesis based on random damage of functional nucleolar organizing regions.