Long-term persistence of chromosome aberrations in uranium miners.

Chromosome aberration analyses were performed on blood samples from 165 active underground uranium miners between 1981 and 1985. After decommissioning the mine in 1997 chromosome aberration analyses were also included in the medical laboratory investigations of health conditions of 141 subjects between 1998 and 2002 within the framework of a follow-up-study. The numerical data are presented as functions of the exposure categories expressed in working level month up to 600. In the active groups the dicentric level was 7 to 12 times higher than in the unexposed population, the acentrics also higher with more than an order of magnitude, the frequency of total aberrations--including dicentrics, acentrics, rings, deletions, minits and numerical aberrations, i.e. both chromatid and chromosome type of aberrations were also well above the control level. In the group of former uranium miners although there were slight decreases in the dicentrics after 8 to 25 yr, the values were not significantly different from the values of active miners. The frequency of deletions was also maintained in the post-mining period. The frequency of acentrics, however, decreased significantly, but even the lowest values remained 2-3 times higher than the values in the unexposed population.The possibility is suggested that for the long-term persistence of cytogenetic alterations the permanent production and presence of clastogenic factors might be responsible. The comparison of the two datasets suggest a long-term persistence of cytogenetic alterations above the population average values in a large fraction of persons investigated.

Role of nitric oxide, cAMP and cGMP in the radiation induced changes of tight junctions in Madin-Darby canine kidney cells.

Radiation induced inflammatory response is thought to be the consequence of acute and chronic oxidative stress, as well as the increased production of various intercellular mediators. Nitric oxide (NO) originated reactive nitrogen species, cGMP and cAMP are well known regulatory factors of the structure and functions of cell contacts. These data raise the possibility that they may play a role in the radiation induced alterations of tight junctions (TJs) and consequently in the radiation injury of surface tissues. Using immunohistochemical methods on confluent cultures of Madin-Darby canine kidney (MDCK) cells, our goal was to clarify the possible role of NO and its relationship with the cGMP and cAMP second messenger systems in the development of the radiation induced alterations of TJs. We found that increased levels of cAMP and/or inhibition of nitrogen oxide synthase (NOS) activity both tend to strengthen TJ associated cell-to-cell contacts in unirradiated control cells. In contrast increased level of cGMP and/or increased expression of NO-sythase, caused the and irregular staining of TJal complexes, which is commonly observed in irridated cells. Our experiments also indicated the protective role of the experimentally increased cAMP level and of NOS inhibitors against the radiation induced TJ changes. All these results suggest the key role of NO in the early radiation response of TJs.

Biological responses of tight junction to ionizing radiation and electromagnetic field expostion.

The tight junctions form and regulate the paracellular barrier in the intercellular spaces between epithelial and endothelial cells. They play important roles in the cellular and pathological processes, which follow exposure to radiation. Therefore, analysis of their changes upon different kind of irradiation may help to understand the basic events governing their function and give important information for the radiobiological research and clinical practice as well. The immunohistochemical data on the distribution of occludin presented here demonstrate the breakdown of tight junctions in Madin Darby kidney cells exposed to ionizing irradiation and show, on the other hand that magnetic field exposures upon 100 microT leave the occludin staining pattern intact.

Radiation responses in plasma membrane. Review of the present state and future trends.

Over the last several decades, the membrane system of the cell has been shown to be a fairly sensitive target for ionizing radiation. As the complex features of membrane functions and structure are revealed more and more, the interest of radiation biology grows. The present review of the biological aspects of ionizing radiation exposure suggests the importance of cell-to-cell contacts through junctions, and the signaling mechanism through receptors.

Did NATO attacks in Yugoslavia cause a detectable environmental effect in Hungary?

Because of the intensive NATO bombardment of the neighboring region to Hungary, i.e., Vojvodina, North Yugoslavia, air monitoring for detection of depleted uranium particles supposed to be used as a component of bullets was extended to the Southern region of the country. Alpha spectrometry was applied as a sensitive analytical technique able to detect uranium. Though no depleted uranium was detected in air by the sensitive technique of alpha-spectrometry, the increased uranium content in natural ratio as a component of normal soil, natural gas, etc., is suggested to originate from well dispersed dust (2.5 microm size) emitted to the atmosphere by explosions during bombing. This observation is supported by the geographical distribution and the relatively rapid decrease of pollution after the bomb attacks ceased.

Effects of mistletoe lectin I and ionizing radiation on the glucose and thymidine uptake in tumour cells in vitro.

The increased uptake of hexose by mammalian cells is considered to be a general response to stress. Nowadays, mistletoe lectin separated from the extracts of the European mistletoe (Viscum album L.) is often used in adjuvant cancer therapy. The present work studies the effect of the lectin on unirradiated and x-irradiated tumour cells. The response of cultured human lung carcinoma cells (Calu-1) was followed by radioactive glucose uptake as well as by tritiated thymidine incorporation. The cells were maintained either in a complete or a so-called restrictive medium. Slight metabolic changes were found in the restrictive medium but not in the complete one. Mistletoe lectin I at a very low concentration (0.001 ng/mL) increased the glucose uptake and thymidine incorporation. Ionizing radiation (1 Gy) did not influence the hexose uptake but it enhanced the incorporation of thymidine. It seems that the actions of two different factors (mistletoe lectin I and radiation) proved to be rather provoking stress effects for the tumour cells as detected in the restrictive medium.

Cellular alterations upon IR-laser (890 nm) exposures, in vivo.

Exposure of cultured cells and small animals to ionizing radiation as well as irradiation of cultured cells with He-Ne laser can cause changes in the functional condition of plasma membranes. The ionizing radiation-induced cell membrane alterations have been determined after either partial or local exposures. The aim of the present study was to reveal whether the local laser treatments cause a general, distant, so called abscopal" effect measured at cellular level, when the laser treatment is intended as a stimulatory procedure. The biological effect of infrared laser (mean power of 5 Watts, 150 Hz frequency, 890 nm wavelength) was demonstrated through 3H-concanavalin A binding by blood cells of daily irradiated (altogether 10 exposures) oncological and non-oncological patients as well as by changes in the proliferation of bone marrow cells of whole body gamma-irradiated (4 Gy) rats, partially laser-treated. The lectin binding of lymphocytes of oncological, as well as ischaemic heart disease patients was increased immediately after the first laser treatment. However, it was decreased after completion of the full course. In cases of inflammatory diseases the test parameters were either unchanged or decreased as compared to their self-control values. The platelets and erythrocytes did not react in any group. Gamma irradiation caused a deep inhibition of proliferation of rat bone marrow cells. The number of fibroblast colony-forming units (CFU-F) could be increased again if the animals were partially exposed to laser. Laser irradiation of one of the femurs led to some recovery of CFU-F values in the exposed as well as unexposed femur. Thus, local infrared laser treatment induces abscopal effects on the cell membrane and cell proliferation characteristics.

X-irradiation-induced changes of the prelysosomal and lysosomal compartments and proteolysis in HT-29 cells.

As a consequence of external and internal ionizing radiation, lysosome-like bodies have been observed to increase both in size and number in some cell types. We investigated this process by morphological methods (electron microscopy, cationized ferritin uptake, acid phosphatase histochemistry, morphometry) in cultured HT-29 cells. In parallel with these studies, we measured the rate of protein degradation on the basis of 14C-valine release from prelabeled cellular proteins. We found that at 2 and 4 Gy doses of X-irradiation the volume of the vacuolar (probably lysosomal) compartment increased without detectable changes of acid phosphatase activity. A 2 Gy irradiation dose did not change protein degradation rate. However, 4 Gy caused a significant inhibition of 14C-valine release from prelabeled proteins. Our results indicate, that the radiation induced expansion of the lysosomal compartment is not necessarily accompanied by increased lytic activity of HT-29 cells.

X-irradiation-induced disorganization of cytoskeletal filaments and cell contacts in HT29 cells.

Organization of cytoskeleton and cell contacts were studied by immunochemistry and electron microscopy in confluent HT29 cultured cells following exposure to 0.5 and 1.0 Gy doses of X-ray. Microtubules were resistant to irradiation, whereas, the actin and intermediate filaments disrupted rapidly following the treatment and their components appeared as clumps of actin and cytokeratin aggregates in the cytoplasm as demonstrated by immunochemistry. Loss of cell contacts and decrease in the number of desmosomes was also characteristic of irradiated cells. Electron microscopy revealed intact desmosomes in control cells and abnormal desmosomes in the irradiated samples characterized by the absence of tonofilaments. The perinuclear filament network and cortical filaments were well detectable by electron microscopy. Under the effect of irradiation, the perinuclear filaments almost disappeared and, at the same time, small bundles of filaments were formed irregularly in the cytoplasm associated with amorphous material.

Effects of modulated microwave and X-ray irradiation on the activity and distribution of Ca(2+)-ATPase in small intestine epithelial cells.

The distribution and activity of Ca(2+)-ATPase were investigated by histochemical methods in small intestine epithelial cells of mice following total body 2450 MHz low frequency (16 Hz) microwave and X-ray irradiation. In the control animals, enzyme activities were found in the brush border and on lateral membranes, including junctional areas of the cells. The enzyme activity of lateral membranes was inhibited by quercetin, a specific inhibitor of Ca(2+)-ATPase. Immediately after square modulated (16 Hz) 2450 MHz microwave irradiation at 1 mW/cm2 power densities, we observed a decreased activity of Ca(2+)-ATPase on the lateral membrane regions. The X-ray irradiation (1 Gy) induced a similar decrease of Ca(2+)-ATPase activity which was reversible within 24 hours. "5 Gy" doses resulted in a decrease of enzyme activities on both apical and lateral membrane areas persisting up to 24 hours following irradiation.

Micronucleus frequency in cultured lymphocytes of an urban population.

The frequencies of micronuclei in cultured cytokinesis-blocked lymphocytes of an urban industrial population of 188 persons were determined. For the mean and SD, 16.0 +/- 7.3 per 1000 CB cells were obtained. A slight but definite age dependence--appr. 0.2/1000 increase per year on average--was indicated by thorough statistical analysis.

Using the micronucleus assay to detect genotoxic effects of metal ions.

The lymphocyte micronucleus assay was used to measure the average frequency of micronuclei in a population and thus assess genotoxic effects. Data from 174 persons give an average value of 16.4 +/- 7.3, and a slight age-dependence was observed. To detect combined environmental mutagen injuries the micronucleus assay was used to study the effects of metal compounds. Cadmium ions increased the micronucleus frequency linearly after incubation with whole blood in vitro with 10(6)-10(-3) M concentrations for 30 min. Similarly, a linear increase in micronucleus frequency was detected with 10(-3)-10(-1) M mercury ions. Concerning the biological effect of selenium, it was found that neither sodium selenite nor selenium dioxide induced increases at concentrations of 10(-7)-10(-6) M; 10(-5) M caused a slight increase; 10(-4) M, however, destroyed the cells. These results suggest that the human lymphocyte micronucleus test can be used to assess genotoxic injuries due to environmental effects in human lymphocytes.

Computerized image analysis for determining micronucleus frequency.

A method for the computerized automation of micronucleus scoring is presented. The task is to identify the cultured, cytokinesis-blocked peripheral lymphocytes (CB cells) and their micronuclei (MN). The main parts of the hardware are the video camera attached to the microscope, the IBM-compatible personal computer with the video digitizer card, and the computer-controlled stage movement unit. The computerized image processing is based on determination and interpretation of contour lines of the CB cells, nuclei, and MN. The BNCTEST image processing software has been developed up to the demonstration phase, and now it has been prepared for the testing period of image series on a large scale.

Morphological and histochemical changes in intercellular junctional complexes in epithelial cells of mouse small intestine upon X-irradiation: changes of ruthenium red permeability and calcium content.

Changes of calcium-content and permeability of tight junction following X-irradiation were investigated in mouse intestinal epithelial cells by electron microscopy. In the control animals the lower parts of tight junctional area as well as the other junctional elements and the intercellular space are labeled by pyroantimonate precipitates, which contain calcium as revealed by electron spectroscopy and electron energy loss spectrometry. X-irradiation, parallel with morphological changes, lead to rapid decrease of pyroantimonate precipitable calcium content and increase of the permeability of tight junctions indicated by the penetration of ruthenium red into the intercellular space. These changes were readily reversible following 0,5 Gy doses of irradiation however, they persisted up to 24 hours following 5 Gy irradiation. We conclude that irradiation at the applied doses can transiently destabilize the tight junctions in the epithelial layer of the small intestine, presumably through a calcium dependent mechanism.

Adenylate cyclase-the more membrane associated, the less radiosensitive.

A dose dependent but not parallel decreases were observed both in SH content and catalytic activity of "free" catalytic subunit after irradiation (0-3200 Gy), while SH groups of membrane-associated adenylate cyclase were insensitive (under 3200 Gy). An initial "radioactivation" of membrane-associated enzyme was found under 800 Gy, then an inhibition above 1600 Gy. The SH alkylating agent, N-ethylmaleimide resulted in a complete inactivation, both of membrane associated form of adenylate cyclase and "free" catalytic subunit with similar inactivation profiles. These data indicate that in the radiosensitivity or "radioprotection" of adenylate cyclase, its membrane association/integration might play a more important role than the SH groups themselves.

Detection of early membrane and nuclear alterations of thymocytes upon in vitro ionizing irradiation.

Some membrane and nuclear parameters of rat thymocytes were studied after in vitro X- or gamma-irradiation with doses of 0.5, 1, 2 and 4 Gy followed by incubation for 0.5 to 4 hours at 21-22 degrees C. Early (within the first 2 hours) distinct functional changes of plasma membranes, i.e. increase in Con A binding, autologous rosette-forming capacity, Alcian Blue-induced agglutination, and a decrease in amount of surface negative charges were observed. Meanwhile, the doses applied did not influence the DNA content, and the proportion of pyknotic nuclei did not grossly differ from that of the time-matched controls. However, an increase in AT-rich DNA component was noted. The radiation-induced changes proved to be transient and dose-dependent. In the whole cell populations no irreversible, death-associated events could be detected under the given experimental conditions.

Modifying effects of the parent and radio-detoxified endotoxins on cell-mediated cytotoxicity and cytokine release.

The cytotoxic effects of the parent and radio-detoxified lipopolysaccharides (LPS and RD-LPS, respectively) were studied at various concentrations (0-50 micrograms/ml) upon cultured human lung carcinoma target cells. There was no significant difference between the effects of the two endotoxins. LPS and RD-LPS at the same concentrations, however, modified the cytolytic activity of human leukocytes (effector cells) and their mediators in different extent. The most remarkable difference was found at 10 micrograms/ml. A much higher cytotoxic activity of effector cells was observed in the case of RD-LPS as compared to the parent LPS. This concentration used for the treatment of X-irradiated and unexposed effector cells at 24 h incubation resulted in elevated release of some cytokines as measured by ELISA. For increasing the natural defence, RD-LPS as immunomodulator may be of practical value.

Effects of low energy beta-irradiation from tritiated water on the morphology of 3T3 fibroblasts.

Cellular alterations of cultured 3T3 cells irradiated with beta-rays from tritiated water were studied by scanning and transmission electron microscopy. We observed decreased negative surface charges, vacuolization of rough endoplasmic reticulum and Golgi-complex, degeneration of mitochondria, increase of lysosomal activity and changes in distribution and amount of microfilaments in the irradiated cells, that parallelled changes in cell shape.

Humoral leukocyte adherence inhibition (H-LAI) follow-up trials on malignant process in mice.

Humoral antitumor response of Lewis lung tumor-bearing mice was measured by humoral leukocyte adherence inhibition (H-LAI) test. The reactivity of serum against tumor antigens prepared from primary tumor and lung metastases could be revealed at the first day after injection of tumor cells. On the other hand, the macroscopical appearance of the primary tumors and lung metastases was observed after 5 and 11 days, respectively. These findings suggest that the immune reaction of the host could be detected by H-LAI test earlier than the tumor can manifest itself. The antigens prepared from primary tumors and metastases seemed to be very similar, however, the metastasis antigen had additional determinants as detected by the H-LAI technique. Comparison of two tumor lines, the original Lewis lung tumor (LLT) and its variant with high metastatic capacity (LLT-HH), showed high similarity between their antigens as tested in H-LAI system by cross-reaction probes.