Dose- and Segment-Dependent Disturbance of Rat Gut by Ionizing Radiation: Impact of Tight Junction Proteins.

The damaging effect of ionizing radiation (IR) exposure results in the disturbance of the gut natural barrier, followed by the development of severe gastrointestinal injury. However, the dose and application segment are known to determine the effects of IR. In this study, we demonstrated the dose- and segment-specificity of tight junction (TJ) alteration in IR-induced gastrointestinal injury in rats. Male Wistar rats were subjected to a total-body X-ray irradiation at doses of 2 or 10 Gy. Isolated jejunum and colon segments were tested in an Ussing chamber 72 h after exposure. In the jejunum, 10-Gy IR dramatically altered transepithelial resistance, short-circuit current and permeability for sodium fluorescein. These changes were accompanied by severe disturbance of histological structure and total rearrangement of TJ content (increased content of claudin-1, -2, -3 and -4; multidirectional changes in tricellulin and occludin). In the colon of 10-Gy irradiated rats, lesions of barrier and transport functions were less pronounced, with only claudin-2 and -4 altered among TJ proteins. The 2-Gy IR did not change electrophysiological characteristics or permeability in the colon or jejunum, although slight alterations in jejunum histology were noted, emphasized with claudin-3 increase. Considering that TJ proteins are critical for maintaining epithelial barrier integrity, these findings may have implications for countermeasures in gastrointestinal acute radiation injury.

Chronic Ouabain Targets Pore-Forming Claudin-2 and Ameliorates Radiation-Induced Damage to the Rat Intestinal Tissue Barrier.

Ionizing radiation (IR) causes disturbances in the functions of the gastrointestinal tract. Given the therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against IR-induced disturbances in the barrier and transport properties of the jejunum and colon of rats. Male Wistar rats were subjected to 6-day intraperitoneal injections of vehicle or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to total-body X-ray irradiation (10 Gy) or a sham irradiation. Isolated tissues were examined 72 h post-irradiation. Electrophysiological characteristics and paracellular permeability for sodium fluorescein were measured in an Ussing chamber. Histological analysis and Western blotting were also performed. In the jejunum tissue, ouabain exposure did not prevent disturbances in transepithelial resistance, paracellular permeability, histological characteristics, as well as changes in the expression of claudin-1, -3, -4, tricellulin, and caspase-3 induced by IR. However, ouabain prevented overexpression of occludin and the pore-forming claudin-2. In the colon tissue, ouabain prevented electrophysiological disturbances and claudin-2 overexpression. These observations may reveal a mechanism by which circulating ouabain maintains tight junction integrity under IR-induced intestinal dysfunction.

Short-Term Mild Hypoxia Modulates Na,K-ATPase to Maintain Membrane Electrogenesis in Rat Skeletal Muscle.

The Na,K-ATPase plays an important role in adaptation to hypoxia. Prolonged hypoxia results in loss of skeletal muscle mass, structure, and performance. However, hypoxic preconditioning is known to protect against a variety of functional impairments. In this study, we tested the possibility of mild hypoxia to modulate the Na,K-ATPase and to improve skeletal muscle electrogenesis. The rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Isolated diaphragm and soleus muscles were tested. In the diaphragm muscle, HH increased the α2 Na,K-ATPase isozyme electrogenic activity and stably hyperpolarized the extrajunctional membrane for 24 h. These changes were accompanied by a steady increase in the production of thiobarbituric acid reactive substances as well as a decrease in the serum level of endogenous ouabain, a specific ligand of the Na,K-ATPase. HH also increased the α2 Na,K-ATPase membrane abundance without changing its total protein content; the plasma membrane lipid-ordered phase did not change. In the soleus muscle, HH protected against disuse (hindlimb suspension) induced sarcolemmal depolarization. Considering that the Na,K-ATPase is critical for maintaining skeletal muscle electrogenesis and performance, these findings may have implications for countermeasures in disuse-induced pathology and hypoxic therapy.

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle.

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

Dose and time dependence of functional impairments in rat jejunum following ionizing radiation exposure.

Ionizing radiation causes dramatic change in the transport and barrier functions of the intestine. The degree of radiation damage rate depends primarily on the absorbed dose and post-irradiation time. Variety of experimental protocols providing different time points and doses exist, with the lack of a common approach. In this study, to develop a unified convenient experimental scheme, dose and time dependence of barrier and transport properties of rat jejunum following ionizing radiation exposure were examined. Male Wistar rats were exposed to total body X-ray irradiation (2, 5, or 10 Gy). The control group was subjected to sham irradiation procedure. Samples of rat jejunum were obtained at 24, 48, or 72 h post-irradiation. Transepithelial resistance, short circuit current (Isc ), and paracellular permeability for sodium fluorescein of jejunum samples were measured in an Ussing chamber; a histological examination was also performed. These parameters were significantly disturbed only 72 h after irradiation at a dose of 10 Gy, which was accompanied by loss of crypt and villi, inflammatory infiltrations, and disintegration of enterocytes. This suggests that found experimental point (72 h after 10 Gy exposure) is the most appropriate for future study using rat jejunum as a model.

The Frequency of Lymphocytes Containing Dumbbell-Shaped Nuclei Depends on Ionizing Radiation Dose and Correlates with Appearance of Chromosomal Aberrations.

Nuclear anomalies of different types appear in cells in response to the action of ionizing radiation after the passage of the first mitotic division. In this article, we present the results of the study of the frequency of occurrence of three types of nuclear anomalies ("tailed" nuclei, nucleoplasmic bridges, and dumbbell-shaped nuclei) in vitro in human lymphocytes cultured with cytochalasin B when exposed to X-rays at doses of 0.0, 0.1, 0.2, 0.4, 0.5, 0.7, 1.0, 1.5, and 2.0 Gy. To stop the cell cycle of cultured lymphocytes after the first mitotic division, a cytokinesis block was performed using cytochalasin B. Dose-dependent curves of the occurrence of lymphocytes containing "tailed" nuclei, nucleoplasmic bridges, or dumbbell-shaped nuclei after irradiation have been constructed. At the same time, frequencies of occurrence of chromosomal aberrations (dicentric and ring chromosomes) in the culture of lymphocytes exposed to the same radiation doses were studied. Comparison of the frequencies of occurrence of dicentric and ring chromosomes with frequencies of occurrence of nuclear anomalies allows us to conclude that these nuclear anomalies are formed as a result of chromosomal aberrations arising in lymphocytes under the action of ionizing radiation. More than that, most of the chromosomal aberrations are converted into dumbbell-shaped nuclei in vitro in the culture of lymphocytes in the cytochalasin block.