The effect of gamma irradiation at sub- and supralethal doses on the lipid composition of the mucosa and of the brush border membrane of the small intestine in rats]

A study was made of the amount of cholesterol and its ethers (phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl serine, and phosphatidyl inositol) in mucosa and membrane of the small intestine brush border 24 h after 4 Gy and 2 h after 20 Gy irradiation. No changes in the lipid content of mucosa and membrane of the brush border were noted after 4 Gy irradiation. Exposure to 20 Gy radiation doubled the number of cholesterol ethers and made the number of individual phospholipids and cholesterol increase by nearly 1.5 times. The amount of phosphatidyl serine in the brush border membrane increased by almost 3 times; the concentration of other lipids increased by nearly 1.5 times; cholesterol/phospholipid ratio was unchangeable.     

Brush border intestinal enzymes after multiple daily fractionation

The modifications in brush border enzyme activity of the epithelial cell of the small intestine were studied after multiple daily fractionation (MDF) of 3 Gy X and 3 Gy X 2 X 2 (12 h split). Disaccharase and dipeptidase activities changed in the same way after irradiation. The results show that both total doses caused the three known phases of increase, decrease, and a return to normal. With MDF, activity at the end of irradiation was similar to or greater than that of controls and remained higher longer than a single dose of 8 Gy. However, the return to normal occurred sooner than after a single dose of 8 Gy. After 11 days, circadian oscillations of brush border enzyme activity appeared similar to those of controls in many segments of the intestine, reaching the highest activity during the night and the lowest in the afternoon.     

Early changes in intracellular ATP concentration after 5 Gy irradiation by routine regimen and regimen modified by low doses (0.1+4.9 Gy)]

Comparative studies were made of immediate (within the first minutes) changes in intracellular ATP concentration after irradiation by different regimens. ATP concentration in cells at a stationary phase of growth increased immediately after irradiation with a dose of 5 Gy, reaching its maximum within 30-40 min. Irradiation with the same total dose, by the regimen of 0.1 + 4.9 Gy at a 3-minute interval between the doses, did not cause alterations in the ATP content in cells. It is concluded that the absence of the increase in the parameter under study (ATP) after irradiation by the latter regimen indicates that preirradiation at a dose of 0.1 Gy inhibits the adequate development of an early response to irradiation with a dose of 4.9 Gy.     

Effects of prolonged low-intensity irradiation on organ weight of the male rat reproductive system

The effects of prolonged irradiation at accumulated doses from 0.5 to 6.0 Gy (dose rate 3.03 cGy/day) on reproductive organs' weight (testes, epididymises, seminal vesicles, prostate) of male rats starting from the early ontogenetic period were studied. On the first day after the irradiation with 1.0 Gy dose a significant loss of the weight in testes and epididymises was revealed. This leaded to the marked atrophy of the organs with the increase of the radiation dose. Long-term restoration of the weight of testes and epididimyses was registered. It was not completed during three months after radiation exposure at 2.0 Gy and higher doses for epididimyses and 4.0-6.0 Gy for testes. The respective changes in the seminal vesicles and prostate weight were less pronounced and had more complicated character. However in the distant period (30-90 days of postreatment) after exposure to 2.0 Gy these parameters were noticeably decreased.     

Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes

We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).     

On the response of a cell population to low-dose irradiation

The cell composition of a population of human blood lymphocytes was studied after irradiation at doses of 5 cGy, 1.0 Gy and 5 cGy + 1.0 Gy and the use of a cytokinesis block. The frequencies of uni-, bi- and multinucleate lymphocytes with and without micronuclei (MN) were taken into account. By the standard criterion the frequency of binucleate lymphocytes with MN among binucleate lymphocytes--the donors were characterized as follows: in with reduction of radiosensitivity after irradiation with 5 cGy + 1.0 Gy as compared to the values of radiosensitivity after irradiation with 1.0 Gy only (an adaptive response, AR); in with no change of radiosensitivity after exposure to these doses (no AR); and with an increased ofradiosensitivity after exposure to these doses (syndrome of increased radiosensitivity, IRS). It was found that upon exposure to 1.0 Gy and 5 cGy + 1.0 Gy in some donors with AR, without AR and with IRS the total numbers of damaged cells in the population and the number of binucleate cells with MN were equal. This result calls in question the involvement of the repair mechanism in the alteration of radiosensitivity of lymphocytes in these donors. It was also observed that in the same donors a simultaneous increase (or a decrease in the case of IRS) of the portion of undamaged binucleate cells in the population took place. Our results demonstrate the existence of a new, populational, mechanism involved in the alteration of radiosensitivity after exposure to the adaptive and challenge doses.     

Effect of ionizing radiation on the structure and functional properties of the basolateral membrane of small intestine enterocytes

The structural state and transport properties of basolateral membrane of rat small intestine enterocytes after exposure to X-ray irradiation (0.5; 1.0 and 2.0 Gy) were studied. The substantional suppression of the active Ca(2+)-transport process concomitant to versatile changes of the membrane structure involving the surface sites and intramembrane protein-lipid complexes was revealed one day after irradiation. Taking into account the early obtained data on apical membrane functional disorders these results confirm that ionizing radiation in sublethal doses induces the structure-function modification of enterocyte plasma membrane affecting the function of the small intestine epithelial cells.     

The effect of amino acids and dipeptides on sodium-ion transport in rat enterocytes

Sodium efflux from isolated intestinal epithelial cells was measured during incubation with several different free amino acids and dipeptides. L-Leucine, which is cotransported with sodium across the brush border membrane, significantly stimulated the total sodium efflux and almost all of this increase involved the ouabain-sensitive flux, i.e., the active component. In contrast, glycyl-L-leucine had little or no effect on active sodium efflux either in the presence or absence of 0.1 mM bestatin, a peptide hydrolase inhibitor. A second dipeptide L-carnosine (beta-alanyl-L-histidine) which is poorly hydrolysed by enterocytes also had no effect upon sodium efflux. However, glycylglycine, which has been shown to be cotransported with sodium, did stimulate the ionic efflux. In addition, measurement of sodium uptake by sheets of small intestine showed that glycyl-L-leucine, carnosine and glycyl-L-proline failed to increase the uptake of the ion, while glycylglycine did significantly stimulate sodium uptake. These data indicate that some dipeptides are not cotransported with sodium, while others are. This suggests that there may well be multiple peptide transporters with very different characteristics in the brush border membrane of enterocytes.     

Effect of gamma-radiation on contents of lipid peroxidation products in animal blood

The dynamics of malonic dialdehyde (MD) dependence in the blood serum of Vistar line male rats on the intensity of irradiation after a total single irradiation with 60Co gamma-quantums in 1.0 and 5.0 Gy doses (during 30 days); 9.0 Gy (during 15 days) were studied. The MD contents values both in the norm and in 0.5, 1, 2, 4, 6, 8, 10, 15, 20 and 30 days after irradiation are given. The experimental data demonstrated the different sensitivity of the final LPO parameters to the doses of 1.0, 5.0, 9.0 Gy radiation depending on the dose power, the changes of these parameters being kept for a long time after irradiation.     

The reactivity of the skeletal muscle arterioles of rats to noradrenaline after whole-body gamma irradiation at a dose of 1 Gy]

Male Wistar rats were exposed by total gamma-irradiation at 1 Gy, Reactions of skeletal muscle arterioles and mean arterial pressure on intravenous doses of noradrenaline (0.1, 0.3, 1.0 and 3.0 micrograms/kg) was studied by intravital microscopy in acute experiments 1, 3 and 5-6 days after irradiation. The exposure causes arterial hypotension on day 1 after that as well as marked reduction of spontaneous arteriole vasomotions and decrease of arteriole constrictions at any doses and dates under study. There are no differences of arterial pressure reaction amplitudes in per cent between control and exposed animals at any doses and dates under investigation.     

The cell composition in the lymphocyte population and the radiation adaptive response

Using lymphocytes of 5 healthy individuals the ability to adaptive response (AR), cell composition of population after PHA stimulation, changes in cell composition population after irradiation in the dose of 1.0 Gy and after irradiation in adaptive (0.05 Gy) and challenge (1.0 Gy) doses have been studied. AR observed in 2 of the 5 individuals only. After PHA stimulation the persons with AR have the total amount of cells after mitosis or during mitosis (the number of binucleated cells + the number of multinucleated cells + the whole cells with micronuclei + the number of mitotic cells) on average is higher than in persons without AR. In individuals with AR the linear correlation between the number of binucleated cells with micronuclei (on the 1000 scored binucleated cells) and the part of binucleated cells in the population is observed with coefficients of correlation -0.89 and -0.91. In the humans without AR this correlation is absent. The correlation observed permits to suppose that AR may occur at the expense of not only the decrease in number of damaged lymphocytes, but also the increase in the share of not damaged binucleated cell with the stable number of damaged cells.     

Effect of gamma-radiation on the content of vitamin E in rats

The dynamics of vitamin E dose-dependence in the blood serum of Vistar line male rats after the total single irradiation with 60Co gamma-quantums in 0.2; 0.5; 1.0; 3.0 and 5.0 Gy doses (during 120 days); 7.0 Gy (during 20 days); 9.0 Gy (during 15 days) were studied. The vitamin E content values both in the norm and in 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 30, 45, 65, 90 and 120 days after irradiation are given. The experiment data show that the dose dependence of vitamin E products has wavelike character. The possible causes of the vitamin E level decrease under intake of small and high doses of gamma-irradiation are discussed.     

On the new mechanism of adaptive response

The role of changes in cell composition of population of human blood lymphocytes in the forming of an adaptive response (AR) has been studied. By micronuclei assay and cytokinetic block with cytohalasin B the frequency of mono-, bi- and multinuclear cells with micronuclei (MN) and without MN were determined in the initial population. The same parameters have been studied after exposure of the population to the adaptive (0.05 Gy), challenge (1.0 Gy) doses and to doses 0.05 + 1.0 Gy 5 hours after. 13 from 23 investigated individuals manifested the AR: the decreasing of the ratio of damaged binuclear cells to the all binuclear cells after the adaptive and challenge exposure. It was shown that the ways of an AR forming are different: in 7 of 13 individuals with AR the number of binuclear cells with MN did not decrease but the amount of binuclear undamaged cells increased. The ratio of these parameters enhances but not for the account of cells with MN decreasing. There is the linear correlation between the frequency of cells with MN and the frequency of binuclear cells in population (spontaneous, after irradiation with doses of 0.05, 1.0 and 0.05 + 1.0 Gy) with the coefficient of correlation about -1. These results show the presence of new mechanism of AR forming, which is not connect with the induction of damage repair and rather with the stimulation of cell division. In the another group of individuals the decrease in damaged cells number after irradiation with doses of 0.05 + 1.0 Gy have been observed. Probably the stimulation of repair system occurred to the moment of 1.0 Gy irradiation. Thus, the mechanism of an AR forming depends on the individual properties of organism. The work was suppoted by RFBR grant 03-04-48325a.     

Effects of gamma radiation on hard dental tissues of albino rats: investigation by light microscopy

The present work aims at studying the effect of gamma radiation on the hard dental tissues. Eighty adult male albino rats with weights of about 250 g were used. The rats were irradiated at 0.2, 0.5, 1.0, 2.0, 4.0 and 6.0 Gy whole-body gamma doses. The effects on hard dental tissue samples were investigated after 48 h in histological and ground sections using light microscopy. Areas of acid phosphatase activity were detected using tartrate-resistant acid phosphatase (TRAP) stains. Observation of histological sections revealed disturbance in predentin thickness and odontoblastic layer as the irradiation dose increased. In cementum, widened cementocytes lacunae were occasionally detected even with low irradiated doses. On the other hand, relatively homogenous enamel was detected with darkened areas in enamel surface at doses over than 0.5 Gy. TRAP-positive cells were detected on the surface of the dentin of irradiated groups as well as cementum surface. Minimal detectable changes were observed in ground sections.     

Myosin-1a is critical for normal brush border structure and composition

To develop our understanding of myosin-1a function in vivo, we have created a mouse line null for the myosin-1a gene. Myosin-1a knockout mice demonstrate no overt phenotypes at the whole animal level but exhibit significant perturbations and signs of stress at the cellular level. Among these are defects in microvillar membrane morphology, distinct changes in brush-border organization, loss of numerous cytoskeletal and membrane components from the brush border, and redistribution of intermediate filament proteins into the brush border. We also observed significant ectopic recruitment of another short-tailed class I motor, myosin-1c, into the brush border of knockout enterocytes. This latter finding, a clear demonstration of functional redundancy among vertebrate myosins-I, may account for the lack of a whole animal phenotype. Nevertheless, these results indicate that myosin-1a is a critical multifunctional component of the enterocyte, required for maintaining the normal composition and highly ordered structure of the brush border.     

Bimodal changes in germinability of pea seeds under the influence of low doses of gamma-radiation

Pea seeds (cv. 'Nemchinovskii-85', harvest of 2002, 80%-germination percentage) were exposed to gamma-radiation with doses ranging from 19 cGy to 100 Gy. One week after the irradiation with doses of 19 cGy and 3 Gy. the germination percentage decreased to 58 and 45%, respectively; at doses of 7 and 10 Gy it was 73 and 70% respectively. At greater doses (25, 50, and 100 Gy), germination percentage decreased in proportion. Anomalous changes in seed germination percentage (as a function of irradiation dose) were caused by the redistribution of irradiated seeds between fractions I and II. The measurements of room temperature phosphorescence in air-dry seeds and the phosphorescence of endogenous porphyrines of imbibing seeds showen that the germination decrease after the irradiation with low doses (19 cGy and 3 Gy) was caused by the increase in the number of weak seeds of fraction II, which had high rates of water uptake and suffered from hypoxia under seed coat. Some of these seeds suffocated from hypoxia, and other seeds produced seedlings with morphological defects (such seeds were considered incapable of germination). During storage of seeds irradiated at doses 19 cGy-10 Gy, the recovery of germination percentage (after its initial decrease) was caused by the decrease in seed number in fraction II. The subsequent germination decrease was caused by seed death. The higher was the irradiation dose, the faster were changes in germination percentage during storage of irradiated seeds. Bimodal changes in pea seed germination with the increase of y-irradiation dose has apparently the same origin as the changes in seed germination during accelerated ageing.     

Brush cells of the mouse intestine possess a specialized glycocalyx as revealed by quantitative lectin histochemistry. Further evidence for a sensory function.

Brush cells occur in the epithelium of the small intestine and in various other epithelia of endodermal origin. Ultrastructural and histochemical characteristics suggest that they represent sensory cells. Because the apical membrane of brush cells might be involved in and specialized for (chemo-)receptive functions, we investigated the composition of the glycocalyx and compared it with that of enterocytes. Ultrathin sections of murine small intestine were labeled with a panel of eight lectins. Their binding sites in the brush border and on vesicles of the apical cytoplasm were detected by colloidal gold and quantified using image analysis. The glycocalyx of brush cells contained significantly higher amounts of l-fucose residues than that of enterocytes, as detected by the lectins UEA-I and LTA. In contrast, most of the other lectins bound more avidly to the glycocalyx of enterocytes. The cytoplasmic vesicles closely resembled the apical membrane in their labeling pattern. Quantitation of the brush cells' distribution revealed that the epithelia of the Peyer's patches contained 10-fold higher numbers of brush cells than the small intestinal mucosa distant from lymphoid tissue. We conclude that brush cells possess a glycocalyx with a specialized composition and differ significantly from enterocytes. Because similar peculiarities of the apical membrane have previously been described for sensory cells of the olfactory and gustatory organs, this study provides further evidence in favor of a sensory function of brush cells.     

Critical target and dose and dose-rate responses for the induction of chromosomal instability by ionizing radiation.

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min(-1)). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around approximately 30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the gamma-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min(-1)) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.     

Blood Glutathione as an Index of Radiation-Induced Oxidative Stress in Mice and Humans

The effect of x-rays on GSH and GSSG levels in blood was studied in mice and humans. An HPLC method that we recently developed was applied to accurately determine GSSG levels in blood. The glutathione redox status (GSH/GSSG) decreases after irradiation. This effect is mainly due to an increase in GSSG levels. Mice received single fraction radiotherapy, at total doses of 1.0 to 7.0 Gy. Changes in GSSG in mouse blood can be detected 10 min after irradiation and last for 6 h within a range of 2.0–7.0 Gy. The highest levels of GSSG (20.1 ± 2.9 ), a 4.7-fold increase as compared with controls) in mouse blood are found 2 h after radiation exposure (5 Gy). Breast and lung cancer patients received fractionated radiotherapy at total doses of 50.0 or 60.0 Gy, respectively. GSH/GSSG also decreases in humans in a dose–response fashion. Two reasons may explain the radiation-induced increase in blood GSSG: (a) the reaction of GSH with radiation-induced free radicals resulting in the formation of thyl radicals that react to produce GSSG; and (b) an increase of GSSG release from different organs (e.g., the liver) into the blood. Our results indicate that the glutathione redox ratio in blood can be used as an index of radiation-induced oxidative stress. © 1997 Elsevier Science Inc.     

Characteristic cytogenetic effects of small doses of ionizing radiation

A study was made of the frequency of chromosome aberrations in human lymphocyte culture after gamma-irradiation (60Co) with doses ranging from 0.05 to 1.0 Gy at dose--rates of 0,005, 0.05 and 0.5 Gy/min. The frequency of structural changes in chromosomes at low doses was higher than it was expected in the case of extrapolating the effect produced by high to low doses of radiation; within the dose range from 0.1 to 0.5 Gy a plateau was registered for aberrations of the exchange type (dicentrics and rings). The abnormal character of the dose dependence of the yield of chromosome aberrations persisted with all three dose - rates under study.