Female reproductive toxicology of cadmium.

The aim of this study was to determine effects of Cd on the structure of ovary, oviduct and uterus after an experimental administration. Animals were divided into three groups. In group A rabbits received cadmium i.p. and were killed after 48 h. In group C Cd was administered p.o. for 5 month. The group K was the control. Decreased relative volume of growing follicles and increased stroma after Cd administration were detected. The number of atretic follicles was significantly higher after administration of Cd. The most frequent ultrastructural alterations observed were undulation of external nuclear membrane, dilatation of perinuclear cistern and endoplasmic reticulum. In all studied types of cells mitochondria with altered structure were found. In the oviduct the highest amount of epithelium in the group with long-term Cd administration was found. Microscopic analysis showed oedematization of the oviduct tissue, caused by disintegration of the capillary wall. An electron microscopic analysis showed dilatation of perinuclear cistern. The intercellular spaces were enlarged and junctions between cells were affected. Mainly after a long-term cadmium administration nuclear chromatin disintegration was present. In the uterus a significant change was determined in the relative volume of glandular epithelium. Increase of stroma was a sign of uterus oedamatization caused by damage in the wall of blood vessels and subsequent diapedesis. After Cd administration alteration in uterus were less expressed, in comparison with ovary and oviduct. Alteration of nuclear chromatin contain following Cd administration suggests degenerative functional changes.

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.

Morphological aspects of ionizing radiation response of small intestine.

Knowledge of the acute and late ionizing radiation exposure damage to the gastrointestinal tract, particularly injury of the small intestine, is of great significance in radiotherapy, as is management of accidental radiation exposure. Irradiation (X-ray, neutron, cobalt gamma) induces a series of events in this rapidly renewing tissue resulting in the well-known symptoms of the gastrointestinal (GI) radiation syndrome, such as GI haemorrhage, endotoxemia, bacterial infection, anorexia, nausea, vomiting, diarrhoea, and loss of electrolytes and fluid. In spite of the significant advances that have occurred in research on underlying mechanisms over the last two decades, the overall etiology and pathogenesis of the GI-syndrome still remains unclear. Currently, to our knowledge, these symptoms are probably due to a rapid modification of the intestinal motility and to the structural alteration of the intestinal mucosa (cell loss and altered crypt integrity). Several evidences suggest that radiation-induced dysfunctions and structural changes of this organ (either changes in subcellular, cellular, and histological structure) are mediated by concerted and interrelated changes of a plethora of various extracellular mediators and their intracellular messengers. The aim of this review is to summarize our current knowledge about the pathomorphology and cell biology of the ionizing radiation response of the GI tract with a focus on the small intestine.

Tight junctional changes upon microwave and x-ray irradiation.

Tight junctions (zonulae occludentes, ZO) are cellularly regulated dynamic structures sensitive to environmental stress agents including ionizing radiation. Radiation induced pathological alterations of the small intestine (gastrointestinal radiation syndrome) are related to altered ZO-mediated paracellular transport. We carried out a quantitative morphological evaluation of the murine jejunal epithelial tight junctional structure in freeze fracture replicas as changed upon whole body X-ray irradiation and low energy microwave exposition. X-ray treatment (4 Gy, 1, 24 h) brought about a partial dearrangement of the ZO strand network which regenerated only partially by 24 h. This observation is in line with data on paracellular permeability increases and ZO-bound calcium drop caused by X-ray irradiation. On the other hand, microwave treatment (16 Hz-modulated 2.45 GHz wave, 1 mW/cm2 power density, I h exposition, samples at I and 3 h after exposition) did not cause dearrangement but, rather an increase in the integration of thight junctional structure, which is in agreement with an increase in cytochemically detectable ZO-bound calcium.

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.

Radiation response of cell organelles.

The cellular responses to various form of radiation, including ionizing- and UV-irradiation or exposure to electromagnetic fields is manifested as irreversible and reversible structural and functional changes to cells and cell organelles. Moreover, beside the morphological signs related to cell death, there are several reversible alterations in the structure of different cell organelles. The radiation-induced changes in the supramolecular organization of the membranes, including plasma membrane, and different cell organelle membranes, play a significant role in the development of acute radiation injury. These signs of radiation-induced reversible perturbation biological membranes reflect changes in the organization and/or composition of the glycocalix, modified activity and/or distribution of different membrane domains, including enzymes and binding sites. The observed changes of the cell surface micromorphology and the alteration of intercellular connections are closely related to the reorganization of the cytoskeletal elements in the irradiated cells. The mitochondria, endoplasmic reticulum, Golgi-complex, the lysosomal system have long been considered to be direct intracellular targets of irradiation. The listed morphological alterations of nuclear chromatin (e.g. changes of fine structure, altered number of nucleolar organizing regions and micronuclei, development of chromosome aberrations) may originate from the radiation-induced damage to the supramolecular organization of DNA and/or nucleus specific proteins. These endpoints of radiation effects resulted as direct consequence(s) of absorbed radiation energy, and indirectly altered intra-, intercellular communication or modified signal transduction. Some complementary data suggest that all these effects are not strictly specific to radiation and may be best considered as general stress responses, similar to those observed after application of various injurious agents and treatments to cells. Moreover, they may be equally responsible for direct degradation of supramolecular component of cells, altered signal transduction, or changes in the amount or ratio of any extracellular mediators upon irradiation. Nevertheless, qualitative and/or quantitative evaluation of any changes of chromosomes by different techniques (morphological analysis of metaphase chromosomes, fluorescent in situ hybridization, development of micronuclei etc.) are useful biological indicators as well as "biological dosimeters" of radiation injury. It is suggested, that some modern methods such as immunohistochemical detection of different proteins, specific markers of cell organelles and cytoskeleton, inspection of distribution of cell surface charged sites and different membrane domains and application of tracer substances may all be included into protocols for evaluation of cell alterations induced by different types and intensities of radiation.

Qualitative enzyme histochemistry and microanalysis reveals changes in ultrastructural distribution of calcium and calcium-activated ATPases after microwave irradiation of the medial habenula.

The localization of calcium and calcium-activated ATPases was investigated electron microscopically in the medial habenula of mice after whole body irradiation with modulated microwaves. In non-irradiated animals calcium-containing precipitates were seen in different subcellular compartments and were often localized on the luminal side of membranes of synaptic vesicles in nerve terminals. At 1 h after 16-Hz modulated microwave irradiation, the number of synaptic vesicles containing calcium precipitates decreased, and reaction products appeared at new locations: in the synaptic clefts and on non-synaptic surfaces of the neuronal plasma membrane. This modified calcium distribution remained unchanged for 24 h following irradiation. Calcium-activated "ecto"-localized ATPase was detected as a punctuated-linear distribution of the reaction product outlining whole areas of glial and neuronal plasma membrane in the habenula of control animals. This pattern did not change on microwave irradiation. However, a quercetin-sensitive "endo"-localized Ca(2+)-ATPase activity appeared in some nerve terminals 24 h after irradiation. Thus, microwave irradiation can influence neuronal calcium homeostasis by inducing Ca2+ redistribution across the plasma membrane and by modifying Ca(2+)-ATPase activity. However, no direct correlation between these effects could be demonstrated by the present study.

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.

Effects of modulated and continuous microwave irradiation on pyroantimonate precipitable calcium content in junctional complex of mouse small intestine.

The pyroantimonate precipitable calcium content of intestinal epithelial cells was investigated in mice following total body irradiation with 2450 MHz continuous and low frequency (16 Hz) square modulated waves. In the control animals the reaction products appeared in the intercellular space of adjacent cells including intermediate junctions and desmosomes and were absent in the area of tight junctions. Immediately after low frequency modulated microwave irradiation at 0.5 and 1mW/cm2 power densities, a rapid distribution of pyroantimonate precipitable calcium content was observed. The pyroantimonate deposits were located on the cytoplasmic side of lateral membrane, in the area of junctional complex, including tight junction, and in other parts of lateral plasma membrane. These changes were reversible and 24 hours after the irradiation the distribution of pyroantimonate deposits was similar to the control. Continuous waves with same energy not altered the distribution of precipitable calcium. We conclude the low frequency modulated microwave irradiation can modify the calcium distribution without heat effects.

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.

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.

Effects of modulated and continuous microwave irradiation on the morphology and cell surface negative charge of 3T3 fibroblasts.

Mouse embryo 3T3 cells were irradiated with 2450 MHz continuous and low frequency (16 Hz) square modulated waves of absorbed energy ranging from 0.0024 to 2.4 mW/g. The low frequency modulated microwave irradiation yielded more morphological cell changes than did the continuous microwave fields of the same intensity. The amount of free negative charges (cationized ferritin binding) on cell surfaces decreased following irradiation by modulated waves but remained unchanged under the effect of a continuous field of the same dose. Modulated waves of 0.024 mW/g dose increased the ruffling activity of the cells, and caused ultrastructural alteration in the cytoplasm. Similar effects were experienced by continuous waves at higher (0.24 and 2.4 mW/g) doses.

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.

Electron microscopic studies on radiation-induced changes of cell surface negative charges.

The amount and distribution of negatively charged sites of different cells (human fibroblasts, B16 melanoma cells, a human lymphoid leukemic cell type) were investigated. In the non-irradiated fibroblasts and B16 melanoma cells the negatively charged sites were localized mainly on apical and lateral surfaces as well as in a polarized manner. However, negatively charged sites on the control human lymphoid leukemic cells often have patched distribution. It was demonstrated that the amount and distribution of negatively charged sites on primary human fibroblasts and B16 melanoma cells changed upon ionizing radiation. However, the topology of negative charges on investigated human leukemic cell membrane did not change.

Surface heterogeneity of tumor cells and changes upon ionizing radiation.

Heterogeneous distribution of surface domains is a characteristic feature of the tumor cell surface and the distribution differs from that of normal cells. During the malignant transformation the heterogeneity may change or disappear. Cell lines with various metastasizing capacities show different distributions of membrane domains or other differences in membrane or surface organization. We have demonstrated that the amount and distribution of negatively charged sites of B 16 melanoma membranes changed upon ionizing radiation (X-ray, 60Co-gamma). In the case of the P 388 lymphoma, however, only the amount of negatively charged sites change after irradiation, the distribution remains unaltered. Both features proved to be radioresistant in human lymphoid leukemic cells.

Polarized membrane domains of fibroblasts.

Data on the heterogeneous distribution of various membrane domains on the surface of fibroblasts are reviewed. Polarized localization of negative charges is demonstrated and new results on the development and maintenance of polarity in primary human fibroblasts are presented. Cell membrane heterogeneity in fibroblasts and in other cells is compared. Our results indicate that the regional localization of membrane domains of fibroblasts, and their dependence on cell movement and cell contacts is in several aspects similar to that described for epithelial cells.

Effect of X-irradiation on adenylate cyclase activity and cyclic AMP content of primary human fibroblasts.

Ionizing radiation provokes an increase of the cAMP level in several organs and body fluids. After reviewing the relevant literature we present the results of our own experiments on primary human fibroblasts. X-irradiation at doses of 0.5 and 2.5 Gy in vitro evoked a rapid and reversible increase of adenylate cyclase enzyme activity. A significant increase in cAMP level of these cells was also observed. Adenylate cyclase was usually localized basolaterally on the surface of unirradiated cells, while irradiation resulted in a modification of distribution, i.e., the enzyme activity also appeared in apical localization.