Activity of some respiratory and lysosomal enzymes of lymphocytes in golden hamster with induced melanoma

A considerable increase in activity of the following enzymes: succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), beta-hydrogenase (beta-HBDH), cytochrome oxidase (CO), and acid phosphatase (AP) was found in lymph nodes lymphocytes after 1 week of malignant melanoma passage in golden hamster (Mesocricetus auratus Waterhouse). The peripheral blood lymphocytes displayed, too, an increase in activity of all the enzymes mentioned except beta-HBDH and SDH. After 3 weeks of the melanoma growth, the animals affected showed a considerable decrease in activity of all the enzymes studied both in the lymph nodes and in the peripheral blood. The increase in enzymatic activity during the initial phase of tumour growth may be a manifestation of biological activation of the cellular defence of lymphocytes. On the other hand, the decrease in the activity seen at the advanced phase of the disease speaks for an impaired immune response.     

Radiosensitivity during the irradiation of animals in an altered gaseous environment. A comparative study on mice and rats of the radiation-protective effect of oxygen-deficient gas mixtures

The radioprotective effect of gas hypoxic mixtures containing 5, 7, 8, 10 and 15% of oxygen on mice and rats was comparatively studied. The dependence of DMF upon oxygen concentration in the mixture was approximated by a hyperbolic function similar to the dependence of the radiomodifying effect of circulatory hypoxia caused by radioprotective agents of the indolylalkylamine series.     

Ultrastructural changes in the neurons of the cerebral cortex of ondatra in acute hypoxia

The reaction of cells in the sensomotor cortex of the ondatra cerebrum has been studied electron microscopically under acute hypoxia conditions. Certain changes have been revealed in neuronal bodies, dendrites, dendritic spines, as well as in synaptic contacts. Reaction of glial cells is noted. The degree of manifestation of the changes essentially varies in different neurons. In most cells, however, they are of reversible character, despite duration of the hypoxia effect. This is evidently connected with the fact, that the ondatra is a diving animal, which is evolutionary adapted to a periodical deficiency of oxygen. The neuronal changes revealed, nevertheless, are not the signs, specifically caused by hypoxia, they are peculiar for many other effects (deafferentation, influences of neurotoxins, stress etc.).     

Radiation protection of mice with hypoxic gas mixtures after administration of anti-hypoxic agents

It is shown that such substances as gutimine, antizol and mexamine increases the resistance of animals to short-term breathing of gas mixtures containing 6 and 5% oxygen. Even if some of them decrease the degree of radioprotective effect of hypoxia, they afford the possibility to safe use of breathing mixtures with lower oxygen content than endured by intact animals, with the resulting increase in radioprotection. Thus the antihypoxic substances can be tested during hypoxiradiotherapy of human tumors.     

The radioprotective effect of hypothermia on the immune and hematopoietic systems in mammals

The functional activity of the synthetic apparatus (parameter alpha) in blood lymphocytes, bone marrow hemopoietic cells, and thymus cells, as well as the total number of blood and bone marrow cells in rats after y-irradiation at a dose of 8 Gy in the conditions of normothermia and hypothermia (16-18 degrees C) with hypoxia-hypercapnia were investigated after 2 h and on days 1 and 4. The recovery processes in blood in both groups of rats after acute X-irradiation at a dose of 7 Gy for 36 days were analyzed too. Under hypothermia, on days 1-4 after acute gamma-irradiation, a decrease in the synthetic activity in remaining cells and devastation in the hemopoietic system were pronounced to a lesser degree. After X-irradiation, the restoration of synthetic activity in blood lymphocytes was shown to begin earlier and to finish faster in "hypothermic" rats as compared with the animals irradiated in the state of normothermia. The survival of "hypothermic" rats was 100% as compared with 30% in "normothermic" animals. Thus, the data show that hypothermia exerts a radioprotective effect on the cells of the immune and hemopoietic systems, thus enhancing the resistance of the organism to radiation.     

Transarterial wall oxygen gradients at the deployment site of an intra-arterial stent in the rabbit

Intimal hyperplasia, common at the deployment site of an intra-arterial stent, may be caused by artery wall hypoxia. The purpose of this study was to determine the effect of an intra-arterial stent on artery wall oxygen concentrations. Transarterial wall oxygen gradients were measured by microelectrode at stent deployment sites in New Zealand White rabbits. Thinned artery walls and decreased oxygen tensions were noted throughout the artery wall immediately following stent deployment with a return toward control values at 28 days. Angioplasty alone had no acute effect on artery wall oxygen concentrations. Larger stent deployment diameters yielded acutely lower artery wall oxygen tensions. Using a linear one-dimensional model for the oxygen profile, we noted that stent deployment resulted in acute changes in oxygen consumption in the inner artery wall that rapidly returned to control values. Changes were noted without differences in blood pressure or arterial blood oxygen concentrations. Oxygen delivery to and consumption within the artery wall are altered by intra-arterial stent deployment. A role for artery wall hypoxia in artery wall pathology at the deployment site of an intra-arterial stent is supported by these findings.     

Enhanced sensitivity of protein kinase B/Akt to insulin in hypoxia is independent of HIF1alpha and promotes cell viability

Maintenance of oxygen homeostasis is a key requirement to ensure normal mammalian cell growth and differentiation. Hypoxia arises when oxygen demand exceeds supply, and is a feature of multiple human diseases including stroke, cancer and renal fibrosis. We have investigated the effect of hypoxia on kidney cells, and observed that insulin-induced cell viability is increased in hypoxia. We have characterized the role of protein kinase B (PKB/Akt) in these cells as a potential mediator of this effect. PKB/Akt activity was increased by low oxygen concentrations in kidney cells, and insulin-stimulated activation of PKB/Akt was stronger, more rapid and more sustained in hypoxia. Reduction of HIF1alpha levels using antimycin-A or siRNA targeting HIF1alpha did not affect PKB/Akt activation in hypoxia. Pharmacologic stabilization of HIF1alpha independent of hypoxia did not increase insulin-stimulated PKB/Akt activation. Although increased insulin-stimulated cell viability was observed in hypoxia, no differences in the degree of insulin-stimulated glucose uptake were observed in L6 muscle cells in hypoxia compared to normoxia. Thus, PKB/Akt may regulate specific cellular responses to growth factors such as insulin under adverse conditions such as hypoxia.     

Ubiquinone-binding site mutations in the Saccharomyces cerevisiae succinate dehydrogenase generate superoxide and lead to the accumulation of succinate

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo.We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.     

Activation of Respiratory Chain Complex II as a Hypoxia Tolerance Indicator during Acute Hypoxia

Activation of respiratory chain complex II during acute hypoxia is an adaptive response that facilitates electron transfer in the respiratory chain when complex I is blocked. Stress induced by acute oxygen deficiency in the body stimulates epinephrine and norepinephrine release into the bloodstream. As a result, compensatory metabolic flows and succinate dehydrogenase and succinate oxidation are activated in the cell. Succinate dehydrogenase activation associated with acute hypoxia exhibits characteristic fluctuations; moreover, stronger stimulation results in oscillations with a shorter period and a higher amplitude. These fluctuations are a consequence of the reciprocal relationship between the sympathetic and parasympathetic systems. In subjects who developed adaptation to hypoxia following repeated sessions of breathing a hypoxic gas mixture, no activation of the succinate–ubiquinone-reductase shunt under hypoxic load was observed. The blood lymphocyte reaction can serve as an indicator of tolerance to acute hypoxia.     

Interaction of cultured mammalian cells with WR-2721 and its thiol, WR-1065: implications for mechanisms of radioprotection

An isothermal microcalorimeter was used to measure changes in heat flow when radioprotective drugs were added to cultured mammalian cells. The heat produced when WR-2721 was added continued for at least 90 min. WR-2721 was dephosphorylated by the cells to thiol (WR-1065) which oxidizes to disulphide. In the microcalorimeter, thiols give an immediate burst of heat due to this oxidation. A biological oxygen monitor revealed that WR-1065 and cysteamine rapidly consumed all the oxygen in culture medium. (10 mM WR-1065 deoxygenated medium in 2 min.) Rapid consumption of oxygen by radioprotective thiols indicates that they will not co-exist with oxygen for long in cells. This has two important implications with respect to mechanisms of radioprotection: (1) oxygen in tissues will be consumed rapidly and could result in local hypoxia; and, (2) at modest doses of protective agents the thiol will be consumed in oxic cells and hence very little will be available for reactions such as hydrogen donation. Our results indicate that anoxia is probably the principal mechanism of protection by aminothiols in mammals and aerated cells. This has major implications for clinical applications of radioprotectors and these are discussed.     

Polymerized bovine hemoglobin decreases oxygen delivery during normoxia and acute hypoxia in the rat

Hemoglobin-based oxygen carriers (HBOC) have been primarily studied for blood loss treatment. More recently infusions of HBOC in euvolemic subjects have been proposed for a wide variety of potential therapies in which increased tissue oxygenation would be beneficial. However, compared with the exchange transfusion models to study blood loss, less is known about HBOC oxygen delivery and vasoacitvity when it is infused in euvolemic subjects. We hypothesized that HBOC [polymerized bovine hemoglobin (PBvHb)] infusion creating hypervolemia would increase oxygen delivery to tissues during acute global hypoxia. Vascular oxygen content and hemodynamics were determined after euvolemic rats were infused with 3 ml of either lactated Ringer or PBvHb solution (13 g/dl, 1.3 g/kg) during acute hypoxia (FIO2 = 10%, 4 h) or normoxia (FIO2 = 21%) exposure. Our data demonstrated that compared with Ringer-infused animals, in hypoxia and normoxia, PBvHb treatment improved oxygen content but raised mean arterial pressure, lowered stroke volume, heart rate, and cardiac index, which resulted in a net reduction in blood flow and oxygen delivery to the tissues. The PBvHb vasoactive effect was similar in magnitude and direction as to the Ringer-infused animals treated with a nitric oxide synthase inhibitor nitro-l-arginine, suggesting the PBvHb effect is mediated via nitric oxide scavenging. We conclude that infusion of PBvHb is not likely to be useful in treating global hypoxia under these conditions.     

Peculiarities of modification by astaxanthin of radiation-induced damages in the genome of human blood lymphocytes exposed in vitro on different stages of the mitotic cycle

The features of astaxanthin impact (20 μg/mL) in the culture of human blood lymphocytes exposed to γ-radiation (1.0 Gy) on the G₀, S, and G₂ phases of the cell cycle were studied using Comet assay. Decrease in the level of DNA damages (Tail Moment index) under astaxanthin influence on lymphocytes irradiated in all stages of cell division was established, while, as a result of previous cytogenetic investigations, lack of the modifying action of astaxanthin after irradiation of cells in the G₂ stage and radioprotective effect in the G0 stage of the mitotic cycle had been revealed. In G₀ phase, the activation of the processes of apoptosis by astaxanthin in irradiated cells with high levels of genomic damages was found. The obtained data demonstrate that astaxanthin has a powerful radioprotective potential, mainly due to its apoptogenic properties.     

Reduced bone marrow pO2 following treatment with radioprotective drugs

The sensitizer adduct technique [( 3H]misonidazole binding) was used to assess the extent of murine bone marrow hypoxia following treatment with a variety of radioprotectors. The binding rates previously determined in vivo were compared to those obtained by incubating marrow cells in atmospheres of varying oxygen content. Parallel experiments demonstrated that the oxygen dependence of [3H]misonidazole binding (Km approximately 0.15% oxygen) was similar to the oxygen dependence of marrow radiosensitivity (Km approximately 0.2% oxygen). Maximally radioprotective doses of several drugs have been shown to increase the binding of [3H]misonidazole significantly in vivo. A comparison to the in vitro binding rates suggests that the average oxygen concentration in the marrow at times associated with radioprotection was on the order of 0.5 to 0.8% oxygen. The relative importance of marrow hypoxia to the overall radioprotective effects of different drugs may vary considerably. However, these results have demonstrated that certain radioprotective drugs can induce marrow hypoxia and this reduced pO2 may contribute to the efficacy of these agents.     

Change in the succinate dehydrogenase activity of peripheral lymphocytes in the rat in early periods following exposure to ionizing radiation

Activity of succinate dehydrogenase (SDH) in peripheral blood lymphocytes of albino mongrel rats was determined during an hour following gamma-irradiation. The highest SDH activity in blood lymphocytes (a 41 per cent increase) was registered 20-30 min following 75 Gy irradiation of the head. With a dose of 20 Gy the SDH response was twice as low. During 1 h following irradiation with a dose of 200 Gy the SDH activity exhibited a 10 per cent decrease.     

GENETIC VARIATION IN HIF SIGNALING UNDERLIES QUANTITATIVE VARIATION IN PHYSIOLOGICAL AND LIFE‐HISTORY TRAITS WITHIN LOWLAND BUTTERFLY POPULATIONS

Oxygen conductance to the tissues determines aerobic metabolic performance in most eukaryotes but has cost/benefit tradeoffs. Here we examine in lowland populations of a butterfly a genetic polymorphism affecting oxygen conductance via the hypoxia‐inducible factor (HIF) pathway, which senses intracellular oxygen and controls the development of oxygen delivery networks. Genetically distinct clades of Glanville fritillary (Melitaea cinxia) across a continental scale maintain, at intermediate frequencies, alleles in a metabolic enzyme (succinate dehydrogenase, SDH) that regulates HIF‐1α. One Sdhd allele was associated with reduced SDH activity rate, twofold greater cross‐sectional area of tracheoles in flight muscle, and better flight performance. Butterflies with less tracheal development had greater post‐flight hypoxia signaling, swollen & disrupted mitochondria, and accelerated aging of flight metabolic performance. Allelic associations with metabolic and aging phenotypes were replicated in samples from different clades. Experimentally elevated succinate in pupae increased the abundance of HIF‐1α and expression of genes responsive to HIF activation, including tracheal morphogenesis genes. These results indicate that the hypoxia inducible pathway, even in lowland populations, can be an important axis for genetic variation underlying intraspecific differences in oxygen delivery, physiological performance, and life history.     

p66Shc is involved in promoting HIF-1alpha accumulation and cell death in hypoxic T cells

Hypoxia results in adaptationally appropriate alterations of gene expression through the activation of hypoxia-inducible factor (HIF)-1 to overcome any shortage of oxygen. Peripheral blood mononuclear cells may be exposed to low oxygen tensions for different times as they migrate between blood and various tissues. We and others have previously shown that T-cell adaptation to hypoxia is characterized by a modulation of cytokine expression and an inhibition of T-cell activation. We have recently demonstrated that the adaptor protein p66Shc negatively regulates T-cell activation and survival. We here show that hypoxia enhances HIF-1alpha accumulation and vascular endothelial growth factor production in T cells. Hypoxic T cells expressed high levels of p21(WAF1/CIP1), of the pro-apoptotic molecules BNIP3, a classic HIF target gene, and BAX, as well as low levels of the anti-apoptotic molecule BCLxl, associated with an induction of cell death. We found out that hypoxic T cells expressed p66Shc. Furthermore, using T-cell transfectants expressing p66Shc, as well as T cells derived from mice p66Shc-/-, we defined a role of p66Shc in T-cell responses to hypoxia. Of interest, hypoxic p66Shc-positive transfectants expressed higher level of HIF-1alpha than negative controls. Thus, p66Shc may play an important role in downstream hypoxic signaling, involving HIF-1alpha protein accumulation and cell death in T lymphocytes.     

Evaluation of the radioprotective effect of total gaseous hypoxia based on the criteria of delayed radiation damage

Breathing with gas mixture containing 6-8% of oxygen during whole-body and large-field irradiation of mice diminishes the radiation-induced life-span shortening (DMF = 2.2) and decreases the degree of canescence (DMF = 1.5), the frequency and severity of nephro- and cardiosclerosis (DMF = 1.5), and the probability of occurrence of pleura and pericardium edemas provoked by cardiopulmonary insufficiency (DMF = 1.4). The fact that hypoxic hypoxia retains its high radioprotective efficiency in relation to the delayed effects of radiation is important for radiotherapy of tumors.     

Radioprotective effect of a herbal product soma

A study was undertaken to examine the functional activity of the synthetic apparatus of rat blood lymphocytes by fluorescence microspectroscopy and the metabolic indices of the animals during the use of the herbal product Soma in normalcy and in acute x-irradiation. It was shown that Soma in normalcy caused a reliable increase in the synthetic activity of the cells with respect to the reference value on the 13th and 20th days of administration. Prior use of Soma for a month with subsequent pause increased the radioresistance of the animal organism (scheme 1), whereas the use of Soma immediately after irradiation (scheme 2) had no appreciable radioprotective effect. The study showed the effect of Soma on enhancement of metabolism, which may play an important role in restoration of the homeostasis of the organism. The results obtained suggest the expediency of further investigation of the radioprotective properties of Soma using various concentrations and administration schemes.     

Nitric oxide (NO) in normal and hypoxic vascular regulation of the spiny dogfish, Squalus acanthias

Nitric oxide (NO) is a potent vasodilator in terrestrial vertebrates, but whether vascular endothelial-derived NO plays a role in vascular regulation in fish remains controversial. To explore this issue, a study was made of spiny dogfish sharks (Squalus acanthias) in normoxia and acute hypoxia (60 min exposure to seawater equilibrated with 3% oxygen) with various agents known to alter NO metabolism or availability. In normoxia, nitroprusside (a NO donor) reduced blood pressure by 20%, establishing that vascular smooth muscle responds to NO. L-arginine, the substrate for NO synthase, had no hemodynamic effect. Acetylcholine, which stimulates endothelial NO and prostaglandin production in mammals, reduced blood pressure, but also caused marked bradycardia. L-NAME, an inhibitor of all NO synthases, caused a small 10% rise in blood pressure, but cell-free hemoglobin (a potent NO scavenger and hypertensive agent in mammals) had no effect. Acute hypoxia caused a 15% fall in blood pressure, which was blocked by L-NAME and cell-free hemoglobin. Serum nitrite, a marker of NO production, rose with hypoxia, but not with L-NAME. Results suggest that NO is not an endothelial-derived vasodilator in the normoxic elasmobranch. The hypertensive effect of L-NAME may represent inhibition of NO production in the CNS and nerves regulating blood pressure. In acute hypoxia, there is a rapid up-regulation of vascular NO production that appears to be responsible for hypoxic vasodilation.