Potential role of the membrane in the development of intestinal cellular damage after whole-body gamma irradiation of the rat

Our study emphasizes the effect of gamma irradiation on intestinal cell membrane fluidity and addresses the potential relationships existing between radiation-induced lipoperoxidation, membrane fluidity, and changes in membrane protein activities. Male Wistar rats were exposed to an 8-Gy total body irradiation (60Co source) and studied 1, 4, and 7 days after irradiation (D1, D4, and D7). Membrane enzyme activities and fluorescence anisotropy were determined on small intestinal crude membrane preparations. The supernatants of membrane preparations as well as plasma were used for malonedialdehyde (MDA) quantification. The effect of carbamylcholine on electrical parameters was estimated on distal ileum placed in Ussing chambers. We observed a decrease in fluorescence anisotropy for at least 7 days, an increase in membrane production of MDA at D4, a decrease in membrane enzyme activities at D4, but an amplification of carbamylcholine-induced increase in short-circuit current at D4 and D7. Furthermore, correlations were observed between the 1,6-diphenyl-1,3,5-hexatriene anisotropy coefficient and sucrase activity and between MDA levels and leucine aminopeptidase activity. Thus, total body irradiation induces changes in intestinal membrane fluidity and an increase in lipoperoxidation. These modifications may have an impact on the activity of membrane proteins involved in intestinal function.     

Effect of long-term exposure to low dose gamma-irradiation on the rat thyroid status

The effect of long-term exposure to low-dose external radiation on the rat thyroid status was studied. The experiments were carried out on Wistar female rats. The single doses absorbed were 0.1; 0.25; 0.5 Gy. The rats were irradiated 20 times (5 days x 4 weeks). The animals were decapitated after 1, 30 and 180 days following the last irradiation. Blood serum was assayed for content of thyroxin (T4) and triiodothyronine (T3) radioimmunologically. The liver was spectrophotometrically assayed for thyroid-induced NADP-malatedehydrogenase (NADP-MDH). It was shown that the long-term 0.5-Gy irradiation of the animals induced a decrease in blood T4 and T3 concentrations 1.34-1.71-fold and 1.24-1.43-fold after 1, 30 and 180 days, respectively. The T3 level was diminished most pronouncedly after 1 day, whereas that of T4--after 30 days following the exposure. With the doses of 0.1 and 0.25 Gy absorbed, the T4 and T3 concentration remained unchanged throughout all the periods studied. The activity of NADP-MDH was decreased 1.55-2.46-fold in all the experimental animals, and it was held decreased after 180 days (1.43-1.50-fold) in 0.25- and 0.5-Gy-irradiated groups, which indicates a disturbance in thyroid hormone metabolism in rats exposed chronically to low-dose radiation. After 180 days, the experimental animals experienced an elevation of thyroid gland weight on 15-20%. The thyroid status disturbance seemed to be due to both inhibited T4 and T3 biosynthesis in thyroid and disturbed hormone peripheral metabolism under radiation exposure.     

Behavioural consequences of an 8 Gy total body irradiation in mice: regulation by interleukin-4

The effects of an 8 Gy gamma total body irradiation (TBI) on exploration and locomotion activities as well as temperature were studied in C57BL6/J mice. Survival, body weight, and blood cell counts were also assessed in irradiated mice treated with placebo or interleukin (IL)-4. The efficacy of IL-4 treatment on improvement in exploration activity was evaluated. The study was carried out from 3 h to 30 days following exposure. Our results showed a biphasic response to irradiation concerning the exploration activity of mice. Irradiated mice had reduced activity as early as 3 h after exposure, with recovery of activity within 24 h. The exploration activity again decreased 4 days after irradiation and the recovery occurred slowly after day 17. IL-4 ameliorated the exploration status in mice in both phases. The locomotion activity was studied using a telemetry apparatus. A similar pattern to that of the exploration data was observed, with a minimal activity observed between days 13 and 17. A radiation-induced hypothermia was also noticed over the same time period.     

Alterations of the VIP-stimulated cAMP pathway in rat distal colon after abdominal irradiation

Ionizing radiation induces hyporesponsiveness of rat colonic mucosa to vasoactive intestinal peptide (VIP). Possible mechanisms responsible for this hyporesponsiveness of the cAMP communication pathway in rat colon were investigated. VIP- and forskolin-stimulated short-circuit current (I(sc)) responses were studied after a 10-Gy abdominal irradiation in Ussing chambers as well as in single, isolated crypts. Adenylyl cyclase (AC) activity and VIP receptor characteristics were determined in mucosal membrane preparations. In addition, alterations in crypt morphology were studied. Impaired secretory responses to VIP and forskolin were observed 4 days after irradiation (decrease of 80%). cAMP analog-stimulated I(sc) responses were unchanged. In isolated crypts, VIP- and forskolin-stimulated cAMP accumulation was markedly reduced by 80 and 50%, respectively. VIP-stimulated AC activity and VIP receptor number were decreased in membrane preparations. No major change of cellularity was associated with these functional alterations. In conclusion, the decreased secretory responses to VIP of rat colon are associated with reduced cAMP accumulation, decreased AC activity, and diminution of VIP receptor numbers without a marked decrease of crypt cell number.     

Change in the reaction of the antioxidant system of wheat sprouts after UV-irradiation of seeds

The response of the antioxidant system of sprouts of wheat Triticum aestivum L. to preliminary irradiation of seeds with UV light was studied. The dependence of lipid peroxidation and the extent of antioxidant activity on the duration of irradiation was studied. It was shown that low doses of UV radiation (5-15 min) stimulate the antioxidant protection of green wheat sprouts grown for eight days. Increasing the irradiation time to 30-60 min leads to the inhibition of lipid peroxidation by the antioxidant system. A more prolonged irradiation of seeds with UV light (for 1-6 h) led to an increase in the level of lipid peroxidation in sprouts. However, 1-2-day-old sprouts from seeds irradiated for 5-6 h, adapted themselves to the influence due to the compensatory mechanisms. By the 8th day of germination of preliminarily irradiated seeds, the content of antioxidants and malone dialdehyde returned to the norm. The dynamics of activity of peroxidase in seeds irradiated with low doses of UV light for 30 min was studied. It was found that on the third day of seed germination, a decrease in peroxidase activity followed by its slight increase occurred. The maximum activity of the enzyme in the endosperm was observed on day 5-6, and in roots and green sprouts, on day 3-5 of germination. It was concluded that antioxidants and peroxidase are involved in the compensatory mechanisms of inhibition of free radicals formed upon UV irradiation of seeds.     

Blue light inactivates plasma membrane H(+)-ATPase in pulvinar motor cells of Phaseolus vulgaris L

Unilateral blue light irradiation induces bending of pulvini of Phaseolus vulgaris towards the source of light. The pulvinar bending is caused by a decrease in turgor pressure of motor cells that are irradiated with blue light. Decrease in the turgor pressure is caused by the net efflux of K(+) and counter anions, accompanying membrane depolarization. In the present study the effect of blue light on the activity of plasma membrane H(+)-ATPase was studied in relation to the membrane depolarization. The activity of the plasma membrane H(+)-ATPase was measured using protoplast suspensions prepared from laminar pulvini from primary leaves. A pulse of blue light under continuous red light irradiation induced both a transient increase in the external pH and transient inhibition of the vanadate-sensitive ATPase. Continuous blue light irradiation under continuous red light irradiation induced both a sustained increase in the external pH and sustained inhibition of the vanadate-sensitive ATPase. These results show that blue light inhibits the activity of the plasma membrane H(+)-ATPase. Inactivation of the plasma membrane H(+)-ATPase supports the membrane depolarization induced by the blue light irradiation.     

Reactions of deoxyribonucleotide synthesis system to irradiation and their modification by radioprotectors

The paper covers the problem on reactions of deoxyribonucleotide (dNTP) synthesis system in blood-forming organs of animals induced by irradiation. The synthesis of dNTP is a rate-limiting stage for DNA synthesis. Cellular requirements for dNTP pools during DNA synthesis are related with ensuring of the accuracy of DNA copying during replication and repair. It has been shown that organism defence mechanisms against irradiation include the following stages: 1. The prompt SOS-activation of dNTP synthesis 30 min later after irradiation, playing the important role in protecting of cell's genetic apparatus from damage. 2. The inhibition of dNTP synthesis within 3-24 h after irradiation resulting to the imbalance of four dNTP and the decrease of their pools. As result of that, the abnormal repair is observed due to depurinations, errors of base incorporations and "misrepair". 3. The restore of dNTP synthesis occurred 2 days later after irradiation. The increase of dNTP pools promotes the increase of DNA synthesis rate as well as proliferative activity of cells. Confirming the fact that the alterations in dNTP pools play essential role in the production of DNA lesions became an important step in understanding of the multistage process leading to radioprotection. To get high and balanced pools of dNTP needed for the increase in the volume of repair of DNA lesions the radioprotectors with high efficiency relative to the survival test were used in experiments. They induced the elevated dNTP synthesis in bone marrow and spleen during the time when the irradiation alone caused the essential prolonged suppression of dNTP synthesis as well as DNA and protein synthesis in organs of nonprotected animals. It has been shown that substances with antioxidant and antiradical activity induced the dNTP synthesis, too. In vivo regulatory factors of dNTP synthesis have been studied to elucidate the mechanisms of getting of high and balanced dNTP pools by using of different substances.     

The irradiation consequencees of animals with alloxan-induced diabetes

The effects of external acute irradiation at dose 1.0 Gy on biologic, haematologic and metabolic changes in blood of alloxan-induced diabetic rats were studied. It was found that the deterioration of diabetic animals occurs in different terms after irradiation exposure, resulting in considerable body weight decrease, well-marked hyperglycemia, abrupt falling of leukocytic system parameters, intensification energetic processes of extant lymphocytes, imbalance of lipid metabolism and thyroid state, as well as significant inhibition of 5'-deiodinase activity in liver tissue.     

Induction of external abnormalities in offspring of male mice irradiated with 252Cf neutron.

To assess the genetic effects of fission neutron, the induction of external malformations was studied in F1 fetuses after F0 male mice were irradiated. Male mice of the ICR:MCH strain were irradiated with 252Cf neutron at doses of 0.238, 0.475, 0.95 and 1.9 Gy. They were mated with non-irradiated female mice at 71-120 days after the irradiation. Pregnant females were autopsied on day 18 of gestation and their fetuses were examined for deaths and external abnormalities. No increases of pre- and post-implantation losses were noted at any dose. External abnormalities were observed at rates of 1.40% in the 0.238 Gy, 2.23% in the 0.475 Gy, 3.36% in the 0.95 Gy and 3.26% in the 1.9 Gy groups; the rate in the control group was 1.65%. The dose-response curve was linear up to 0.95 Gy, and then flattened out; the induction rate of external abnormalities was 2.7 x 10(-4)/gamete/cGy based on the linear regression. These results indicated that fission neutron effectively induces external abnormalities in F1 fetuses after spermatogonial irradiation.     

Peculiarities of the ovary structure in the rat offspring developing under conditions of the removed thyroid gland of female rat with the thyroxine replacement therapy and external radiation exposure in utero

The influence of a single external gamma-radiation at a dose of 1.0 Gy (dose rate 9.08 x 10(-4) Gy/sec) on the 15th day of gestation in case of the removed complex of thyroid and parathyroid glands (thyroidparathyroidectomy) on the first day of gestation, as well as introduction of thyroxin and CaC12 on the structure of offspring ovary in postnatal ontogenesis (30-day old animals) was studied. It has been shown that thyroidparathyroidectomy of a female mother rat with thyroxine replacement therapy and irradiation, as well as the combination of these factors disturb the structure of ovarian tissues of the offspring. A single external irradiation on the 15th day of embryogenesis causes death of a considerable part of primordial follicles in the offspring ovary and growth of follicular layers in the secondary follicles. Thyroidparathyroidectomy of female rat on the first day of gestation with thyroxine replacement therapy causes delay in the development of follicles in the ovary at the early stages of maturation of 30-day old animals. The radiosensitivity of the ovarian tissues of the offspring that has been developed under the combined effect of the factors studied increases and results in an almost full loss of pool cells in the ovary of infant rats.     

Effect of gamma irradiation on insulin receptor interaction in rat erythrocytes

An insulin receptor interaction has been studied in rat erythrocytes after whole-body gamma irradiation (1 Gy). Specific binding of insulin was found to increase 30 days following irradiation against the background of a decreased immunoreactive insulin concentration in the blood. A change in the postirradiation activity of insulin receptors is considered as a manifestation of the homeostatic mechanism of "up" regulation in exposed animals.     

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

The late effects of radiation on hematopoietic stem cells and the possibility of their modification]

In experiments with mice it has been found that a radioprotective agent, mexamine (two different forms), administered prior to a whole-body single exposure at a dose of LD0/30 increases the average life of animals and the number of exoCFUs 8 days and has no influence on the number of exoCFUs 12 days. Mexamine does not modify the decrease of haemopoietic colonies in sizes in recipients, mice survived acute radiation sickness being used as donors. The share of CFUs 8 days at the stage of DNA synthesis has been shown to increase with age, as well as in animals which lived for 14 months after irradiation.     

Population dynamics of the murine lymphokine activated killer system: precursor frequency and kinetics of maturation and renewal

Abstract The proliferation kinetics and population renewal of recombinant interleukin-2 (rIL-2)-induced murine lymphokine activated killers (LAK) arising from splenic precursors was studied. Extensive proliferation has been shown to accompany the de novo generation of LAK cytotoxicity. In this report, a thymidine 'hot pulse' suicide technique was employed to examine the sensitivity of LAK progenitors during various time periods following culture initiation. Hot pulse during the first 24 hr of culture resulted in a 30–35% reduction in lytic activity when assayed on day 5. Pulse periods between days 1 and 4 resulted in almost complete inhibition (90–95%) of lytic function when assayed on day 5. Proliferation of LAK progenitors was documented by limiting dilution analysis comparison of splenic precursors and functionally mature LAK cultures. These studies showed a 75- to 80-fold enrichment of LAK progenitors after 3 days culture in rIL-2. By flow cytometric cell cycle analysis, we demonstrated that the number of cells in the S/G₂/M phase increased with the length of rIL-2 culture and represented approximately 40% of the cells by day 4. Finally, we used the rate of decay of lytic activity following irradiation as a factor to define the mean life span of a cytotoxic effector in the absence of cellular input. An exponential decrease to approximately 50% of controls was observed within 8–9 hr after irradiation. Taken together, these results suggest that the LAK system is highly dynamic and requires continuous cellular proliferation for its maintenance.     

Effect of gamma irradiation on immobilized trypsin

The effect of ionizing radiation of 0.05-10 Mrad on trypsin immobilized on dialdehyde cellulose was being studied. After irradiation the activity of native trypsin decreases by 25%, as compared with the initial, while the activity of immobilized trypsin remains constant. Before immobilization cellulose undergoes special pretreatment that leads to a decrease in the initial contamination. Some samples of modified cellulose were contaminated by staphylococcus culture (200,000 microbes per 0.2 g) and then exposed to irradiation of 0.05-0.4 Mrad. A distinct correlation between the irradiation dose (0.05-0.4 Mrad) and contamination of the object was registered.     

Small doses of X-ray irradiation activate the NO-synthase component of the nitric oxide cycle

It has been shown that chronic X-ray irradiation, (CRI), activates the formation of NO in rats. This is apparent in the increase in the level of NO2- in the blood plasma from 12.59 +/- 1.7 to 39.79 +/- 2.9 nmol/ml after 10 days of irradiation. On the 20 and 30 day of CRI, the level of NO2(-)- was 21.05 +/- 1.2 and 30.73 +/- 1.9 nmol/ml respectively. The changes in the NO-synthase component of the NO cycle were accompanied by a decrease in the osmotic resistance of the erythrocytes and the nitritreductase activity of hemoglobin.     

Involvement of DNA-dependent protein kinase in UV-induced replication arrest.

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G(1)/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs(+))), as measured by [(3)H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs(-)) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs(+) cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs(-) cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs(+) cells but not in DNA-PKcs(-) cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs(+) cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs(-) cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation.     

Protection against radiation oxidative damage in mice by Triphala

Protection against whole body gamma-irradiation (WBI) of Swiss mice orally fed with Triphala (TPL), an Ayurvedic formulation, in terms of mortality of irradiated animals as well as DNA damage at cellular level has been investigated. It was found that radiation induced mortality was reduced by 60% in mice fed with TPL (1g/kg body weight/day) orally for 7 days prior to WBI at 7.5 Gy followed by post-irradiation feeding for 7 days. An increase in xanthine oxidoreductase activity and decrease in superoxide dismutase activity was observed in the intestine of mice exposed to WBI, which, however, reverted back to those levels of sham-irradiated controls, when animals were fed with TPL for 7 days prior to irradiation. These data have suggested the prevention of oxidative damage caused by whole body radiation exposure after feeding of animals with TPL. To further understand the mechanisms involved, the magnitude of DNA damage was studied by single cell gel electrophoresis (SCGE) in blood leukocytes and splenocytes obtained from either control animals or those fed with TPL for 7 days followed by irradiation. Compared to irradiated animals without administering TPL, the mean tail length was reduced about three-fold in blood leukocytes of animals fed with TPL prior to irradiation. Although, similar protection was observed in splenocytes of TPL fed animals, the magnitude of prevention of DNA damage was significantly higher than that observed in leukocytes. It has been concluded that TPL protected whole body irradiated mice and TPL induced protection was mediated through inhibition of oxidative damage in cells and organs. TPL seems to have potential to develop into a novel herbal radio-protector for practical applications.     

The composition and physicochemical properties of the blood lipoproteins in rats exposed to external gamma irradiation]

Content of lipids, character of chemiluminescence of blood plasma and certain classes of lipoproteins have been studied. Geometrical parameters, nature and quantity of charged groups of lipoprotein particles accessible for titration have been determined 1 and 30 days after a single external gamma irradiation of rats in a dose of 3 Gy. The used irradiation dose exerts an expressed hyperlipidemic effect retained for one month after irradiation. The disturbances in the spectrum of blood lipids and lipoproteins are of hyper-beta and hyper-prebeta lipoproteinemia character. Considerable disturbances of physicochemical properties of different classes of lipoproteins have been detected. They are exhibited in changes of the pattern of free-radical processes, state of the charge of surface ionogenic groups and geometrical parameters of lipoprotein particles. Changes registered by the methods of potentiometric titration and correlation spectroscopy are most expressed in lipoproteins of very low density and those of low density.