Role of Bioenergy Effects of Cystamine in Realising Potentiation of Radioprotective Properties of the Radioprotector in its Repeated Administration

The findings of the study on the impact of hypoxia on the glow of the Black Sea ctenophore Mnemiopsis leidyi A. Agassiz, 1865 of three size groups (20-30, 30-45, and 45-60 mm) were obtained under experimental conditions. Peculiarities of ctenophore bioluminescence were studied during mechanical and chemical stimulation under the conditions of normoxia (at an oxygen concentration of 5.6-6.7 mg O2 ·L-1 ), moderate hypoxia (2.5-2.8 mg O2 ·L-1 ), and acute hypoxia (1.2-1.5 mg O2 ·L-1 ). An increase in the amplitude and energy of luminescence of the ctenophores mechanically and chemically stimulated was observed at an oxygen concentration of 1.2-1.5 mg O2 ·L-1 (acute hypoxia) in two size groups in the lobate form (30-45 and 45-60 mm). The inhibition of amplitude, energy, and duration of the signal was registered in M. leidyi ctenophores at the transitional stage from larva to the lobate form under conditions of acute hypoxia. It was noted that in normoxia, the values of the amplitude and energy of the bioluminescent signal of M. leidyi increase along with a size growth of an individual. This phenomenon was observed both during mechanical and chemical stimulations. Under conditions of acute hypoxia, this trend was mainly preserved. The universality of the relation between the bioluminescence of the organisms and their bioenergetics is obvious. The bioluminescent system of ctenophores has the role of an antioxidant system and is engaged in the neutralization of reactive oxygen species, that is the process during which photons are emitted. The response of the bioluminescent system to a decrease in О2 concentration can be associated with an increase in the production of ROS that provides high values of the ctenophore luminescence under hypoxic conditions.

Previous work from our laboratory had demonstrated attenuation of systemic vasoreactivity to pressor agents in rats after acute or chronic exposure to hypoxia. Therefore we hypothesized that hemorrhage of acutely hypoxic (12% O2) or chronically hypoxic (barometric pressure 380 mmHg for 3 wk) rats would deter the normal increase in total peripheral resistance (TPR) and thus decrease the ability to maintain blood pressure. Progressive hemorrhage (2% of blood volume per min) was performed under conditions of either normoxia or acute hypoxia in conscious rats. In control animals, the increase in TPR observed during normoxic hemorrhage was absent when hemorrhage was performed in acute hypoxia. Furthermore, the amount of blood removal required to achieve hypotension was reduced under conditions of acute hypoxia. In contrast, chronically hypoxic rats exhibited no difference in the threshold for hypotension between conditions of acute normoxia and hypoxia and demonstrated an increased hypotensive threshold under both normoxic and hypoxic conditions compared with control animals. We next hypothesized that the prolonged threshold for hypotension observed in chronically hypoxic rats might be due to hypoxia-induced right ventricular hypertrophy. Such ventricular hypertrophy may minimize stimulation of ventricular volume receptors thought to elicit the reflex fall in heart rate and TPR occurring in extreme underfill conditions. Therefore we compared the cardiovascular responses to hemorrhage in rats with right ventricular hypertrophy resulting from administration of monocrotaline with those from rats treated with vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)

BackgroundStudies have revealed the protective effect of DL-3-n-butylphthalide (NBP) against diseases associated with ischemic hypoxia. However, the role of NBP in animals with hypobaric hypoxia has not been elucidated. This study investigated the effects of NBP on rodents with acute and chronic hypobaric hypoxia.MethodsSprague-Dwaley rats and Kunming mice administered with NBP (0, 60, 120, and 240 mg/kg for rats and 0, 90, 180, and 360 mg/kg for mice) were placed in a hypobaric hypoxia chamber at 10,000 m and the survival percentages at 30 min were determined. Then, the time and distance to exhaustion of drug-treated rodents were evaluated during treadmill running and motor-driven wheel-track treadmill experiments, conducted at 5800 m for 3 days or 20 days, to evaluate changes in physical functions. The frequency of active escapes and duration of active escapes were also determined for rats in a shuttle-box experiment, conducted at 5800 m for 6 days or 27 days, to evaluate changes in learning and memory function. ATP levels were measured in the gastrocnemius muscle and malonaldehyde (MDA), superoxide dismutase (SOD), hydrogen peroxide (H2O2), glutathione peroxidase (GSH-Px), and lactate were detected in sera of rats, and routine blood tests were also performed.ResultsSurvival analysis at 10,000 m indicated NBP could improve hypoxia tolerance ability. The time and distance to exhaustion for mice (NBP, 90 mg/kg) and time to exhaustion for rats (NBP, 120 and 240 mg/kg) significantly increased under conditions of acute hypoxia compared with control group. NBP treatment also significantly increased the time to exhaustion for rats when exposed to chronic hypoxia. Moreover, 240 mg/kg NBP significantly increased the frequency of active escapes under conditions of acute hypoxia. Furthermore, the levels of MDA and H2O2 decreased but those of SOD and GSH-Px in the sera of rats increased under conditions of acute and chronic hypoxia. Additionally, ATP levels in the gastrocnemius muscle significantly increased, while lactate levels in sera significantly decreased.ConclusionNBP improved physical and learning and memory functions in rodents exposed to acute or chronic hypobaric hypoxia by increasing their anti-oxidative capacity and energy supply.

We studied the effect of injections of melatonin and modifications of the duration of illumination on the activity of 5′-nucleotidase, an enzyme providing synthesis of adenosine, in the forebrain of juvenile male albino rats. The measurements were performed under conditions of acute hypobaric hypoxia. We found that, under conditions of natural illumination, neither isolated injections of melatonin nor acute hypoxia noticeably changed the activity of 5′-nucleotidase. At the same time, acute hypoxia combined with melatonin injections increased the activity of this enzyme. A similar noticeable rise in the activity of 5′-nucleotidase was observed after melatonin injections in normoxic animals kept in constant darkness, and in rats subjected to hypoxia without the above injections but under conditions of constant illumination. These data allow us to suppose that melatonin (whose level in the extracellular medium is a factor providing synchronization of endogenous temporal rhythms) stimulates 5′-nucleotidase-mediated production of adenosine in brain neurons. Acute hypoxia promotes such an effect of melatonin.

We studied the effects of in vivo modulation of activity of mitochondrial ATP-dependent potassium channel (mitoKATP) by uridine on the morphofunctional state of mitochondria in rat cardiomyocytes under conditions of acute hypoxia. Preinjection of uridine to animals reduced the number of structurally modified mitochondria, but had practically no effect on their morphogenesis after hypoxia. Uridine in vivo stimulated the formation of micromitochondria and their release into the cytoplasm. The number of "maternal" mitochondria containing three and more new micromitochondria, increased as well. The use of mitoKATP blocker 5-hydroxydecanoate in parallel with uridine abolished its protective effect, as it significantly inhibited the formation of micromitochondria in rat cardiomyocytes after acute hypoxic exposure.

The transcription factor (TF) HIF1 is one of the most important factors of adaptation to chronic cerebral hypoxia. However, under the conditions of acute hypoxia and reoxygenation, the stress response TF (NRF2) becomes important. The interaction between these proteins at the level of regulation of antioxidant defense and glucose metabolism has been shown previously in hypoxia-sensitive cancer tumors. Here, we have studied the effect of severe hypobaric hypoxia (SH) on HIF1- and NRF2-dependent processes in the rat neocortex. We revealed the joint regulation of glutathione-dependent antioxidant systems by these proteins, which influenced the total antiradical activity and the cellular redox status. In particular, HIF1 inhibition prevented the SH-induced oxidative shift 23 h after reoxygenation, which was accompanied by an increase in the content of total glutathione and the activity of glutathione reductase. Both of these effects were NRF2-dependent, which suggests that this transcription factor is activated in response to SH in combination with HIF1 inhibition. The data confirm the previous hypothesis about the maladaptive effect of HIF1 under the conditions of acute hypoxia and reoxygenation and point to the contribution of NRF2 the protective mechanisms in the post-hypoxic period. The hypothesis of interaction between these transcription factors in the (post)hypoxic period requires further verification and may have substantial influence on understanding the molecular pathomechanisms of cerebral hypoxia.

The article focuses on the treatment and protective effects of hypoxyradiotherapy during external-beam irradiation of cervical carcinoma, including paraaortic lymph nodes, combining radiotherapy with californium-252 (252Cf) neutron brachytherapy. An analysis of treatment results, early and late side effects and complications is presented. From January 1989 to May 1997, 307 women with stage IIb and IIIb cervical carcinoma, treated with 252Cf neutron brachytherapy, were randomly divided into two groups and treated with external-beam irradiation to the paraaortic lymph nodes as follows: • 155 patients (59 with stage IIb, 96 with stage IIIb) were treated by external-beam irradiation administered as a 60-Gy dose applied under conditions of acute hypoxia; 77 patients (30 with stage IIb and 47 with stage IIIb) received extended-field irradiation up to L4 and 78 patients (29 with stage IIb and 49 with stage IIIb) up to T12. • 152 patients (58 with stage IIb, 94 with stage IIIb) were treated by external-beam irradiation administered as a 40-Gy dose applied under normal oxygenation conditions. 73 patients (29 with stage IIb and 44 with stage IIIb) received extended-field irradiaton up to L4 and 79 patients (29 with stage IIb and 50 with stage IIIb) up to T12. The same 56 Gy-equivalent (eq) doses at point A and 19 Gy-eq doses at point B were applied intracavitarily in both groups. The total radiation doses at points A and B were 99 and 79 Gy-eq, respectively, for patients treated with external-beam irradiation to 60 Gy under conditions of acute hypoxia. For patients treated with external-beam irradiation to 40 Gy under normal oxygenation conditions, the doses at points A and B were 85 and 59 Gy-eq, respectively. The 5-year overall survival rate for all patients (stages IIb and IIIb) was 7.0% better for patients treated in acute hypoxia than for patients treated under normal oxygenation conditions (78.7% vs. 71.7% [p < 0.16]). The 5-year metastases-free survival rate was better by 11.7% for stage IIIb patients in the hypoxyradiotherapy group with extended field up to T12 as compared to patients with extended field up to L4 (97.4% vs. 85.7% [p < 0.05]). Comparison of metastases-free survival rate of stage IIIb patients after external-beam irradiation with extended field up to T12 in hypoxic condition versus normoxic condition showed a 12% better result for patients in hypoxic condition (97.4% vs. 85.4% [p < 0.04]). Occurrences of symptomatic radiation-induced reactions during or shortly after irradiation were more frequently observed in patients treated with a lower dose under normoxic conditions. During the period of 6-12 years after treatment there were no changes in the frequencies of occurrences of late effects and complications. The importance of the protective effects of hypoxyradiotherapy for dose escalation in external-beam irradiation of cervical carcinoma, including paraaortic lymph nodes, with regard to an improvement of the cure rates of metastases in paraaortic lymph nodes has been confirmed.

GRDINA, D. J., BASIC, I., GUZZINO, S., AND MASON, K. A. Radiation Response of Cell Populations Irradiated in Situ and Separated from a Fibrosarcoma. Radiat. Res. 66, 634-643 (1976). A cell separation system involving the centrifugation of cells in linear density gradients of Renografin provides a useful method for the in vivo study of the various clonogenic subpopulations comprising a solid tumor. Following this procedure, two selected clonogenic cell populations were separated from a murine fibrosarcoma after exposure in situ to ionizing radiation. The clonogenicity of each population (Band 2, fp = 1.08 g/cm3 and Band 4, # = 1.14 g/cm3) was determined using a lung colony assay. Survival curves were constructed and compared with data from an unseparated population. The Band 2 population, when irradiated in air-breathing animals, had a relatively large fraction of sensitive cells and a more resistant component whose response was apparent at doses greater than 1000 rad. The Do of this resistant fraction was 459 rad. Comparing the survival curves of Band 4 and unseparated cells, the values of Do were similar (403 and 413 rad, respectively), but the parameters describing the shoulder region of these curves differed significantly (Band 4, D, = 644 rad, n = 5.2; and unseparated cells, D, = 344 rad, n = 2.3). Under conditions of acute hypoxia, Band 2 cells became significantly more radioresistant. No change in radiation sensitivity was apparent, however, for Band 4 cells. It was concluded that the Band 4 population probably consisted primarily of chronically hypoxic cells since conditions of acute hypoxia had little, if any, effect on their response to radiation.

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.

Effective communication between multipotent mesenchymal stromal cells (MSCs) and endothelial cells (ECs) plays a critical role in the regulation of angiogenesis, especially under conditions of hypoxia. In addition to paracrine stimulation, direct intercellular contacts play an important role in the angiogenic interaction between MSCs and ECs, making them an important target for modulating vascular network restoration under ischemic conditions. The aim of this study was to determine the contribution of gap junctions (GJs) to the angiogenic response of MSCs and the EA.hy926 cell line (an Endothelial Cell Model) under acute hypoxic stress. In a cell co-culture model at 0.1% O2 using a specific GJ inhibitor (carbenoxolone), molecular, cellular, and functional tests were performed: assessment of viability, proliferation, migration, secretion of angiogenic mediators, and expression of crucial genes. GJ blockade was accompanied by decreases in the proliferation and migration activity and angiogenic potential of the conditioned medium in in vitro and in ovo tests. These data highlight the importance of the GJ in coordinating the angiogenic response in conditions of acute hypoxia and can be used to develop protocols for regenerative medicine.

BackgroundUnder conditions of hypoxia, cancer cells with hypoxia inducible factor-1α (HIF-1α) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1α under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited.MethodsIn this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown.ResultsHere we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1α. We also demonstrated that NADH functions as a signal for conveyance of HIF-1α degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1α via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1α is recogonized by VHL, which leads to degradation of HIF-1α via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1α was independent of proline hydroxylation.ConclusionsOur findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1α stability. These results also demonstrate the involvement of VHL in degradation of HIF-1α through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1α acetylation in hypoxia.

Our objective was to evaluate the effect of conditioning and hypoxia on rectal and gastrointestinal temperature changes in dogs exercising at cold ambient temperature. Six Alaskan Husky sled dogs, each in a physically conditioned and unconditioned state, were used in the prospective study. Dogs in peak physical condition were run untethered on a treadmill under normoxic and hypoxic conditions of 20 and 12.5% environmental oxygen concentration, respectively, on separate days. After undergoing a deconditioning period of four months, the same dogs were run again under the same environmental conditions of 20 and 12.5% O2. Body temperature measurements were obtained via digital rectal thermometer and ingestible gastrointestinal thermistor at baseline, every 5 min for 30 min of exercise, and for 15 min following cessation of exercise. Under hypoxic conditions, peak gastrointestinal temperature was lower in conditioned vs unconditioned dogs. Gastrointestinal cooling was faster in conditioned dogs under normoxic conditions only. There was no difference in the peak rectal temperature, or rate of rectal temperature cooling in either normoxic or hypoxic conditions. 3 of 6 (50%) of the conditioned dogs reached a plateau temperature after approximately 20 to 25 min. This was observed less frequently when the dogs were unconditioned. Gastrointestinal and rectal temperatures reacted differently to exercise depending on conditioning or environmental oxygen concentration and this suggests that they cannot be used interchangeably to assess body temperature. Under these conditions, conditioning had no effect on rectal temperature. Presence of a plateau effect of rectal or gastrointestinal temperature may demonstrate a thermoregulatory balance of heat production and heat loss and may be a helpful measure in assessing individual dogs’ level of conditioning with regard to resistance to thermal stress.

Acute hypoxia increases pulmonary arterial (PA) pressures, though its effect on right ventricular (RV) function is controversial. The objective of this study was to characterize exertional RV performance during acute hypoxia. Ten healthy participants (34±10 years, 7males) completed three visits: visits 1 and 2 included non-invasive normoxic (fraction of inspired oxygen ( )=0.21) and isobaric hypoxic ( =0.12) cardiopulmonary exercise testing (CPET) to determine normoxic/hypoxic maximal oxygen uptake ( ). Visit 3 involved invasive haemodynamic assessments where participants were randomized 1:1 to either Swan-Ganz or conductance catheterization to quantify RV performance via pressure-volume analysis. Arterial oxygen saturation was determined by blood gas analysis from radial arterial catheterization. During visit 3, participants completed invasive submaximal CPET testing at 50% normoxic and again at 50% hypoxic ( =0.12). Median (interquartile range) values for non-invasive values during normoxic and hypoxic testing were 2.98 (2.43, 3.66) l/min and 1.84 (1.62, 2.25) l/min, respectively (P<0.0001). Mean PA pressure increased significantly when transitioning from rest to submaximal exercise during normoxic and hypoxic conditions (P=0.0014). Metrics of RV contractility including preload recruitable stroke work, dP/dtmax, and end-systolic pressure increased significantly during the transition from rest to exercise under normoxic and hypoxic conditions. Ventricular-arterial coupling was maintained during normoxic exercise at 50% . During submaximal exercise at 50% of hypoxic , ventricular-arterial coupling declined but remained within normal limits. In conclusion, resting and exertional RV functions are preserved in response to acute exposure to hypoxia at an =0.12 and the associated increase in PA pressures. KEY POINTS: The healthy right ventricle augments contractility, lusitropy and energetics during periods of increased metabolic demand (e.g. exercise) in acute hypoxic conditions. During submaximal exercise, ventricular-arterial coupling decreases but remains within normal limits, ensuring that cardiac output and systemic perfusion are maintained. These data describe right ventricular physiological responses during submaximal exercise under conditions of acute hypoxia, such as occurs during exposure to high altitude and/or acute hypoxic respiratory failure.

The effect of experimental amyloidosis on the morphology of the Paneth cells of the mouse was investigated by using light and fluorescence microscopy. Amyloidosis was induced by daily subcutaneous casein injections for 4 weeks. It was found that the number of secretory granules of the Paneth cells increased in the jejunum, but remained normal in the duodenum. Small morphological alterations took place in the Paneth cells during the induction of amyloidosis, and occasionally the Paneth cells were totally lost. These changes suggest that the function of the Paneth cells of the jejunum may be partly inhibited during experimental amyloidosis.

Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0-4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins (actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.