Biochemical indices of the sensitivity of cold-adapted animals to hyperoxic exposure

It has been established that white rats adapted to the low temperature of the environment (45 days 2-4 degrees C) in contrast to the animals undergoing such action during 3 days (cold stress) show resistance to the toxic action of the high oxygen pressure. The considerable removal of convulsions under the action of oxygen, the absence of increase of erythrocyte membrane permeability in the preconvulsive stage of hyperoxia and prevention of changes in the substrate specificity of type A monoamine oxidase both in preconvulsive and convulsive periods of oxygen intoxication are their characteristics.     

Monoamine oxidase activity and effect of clorgyline on the polyamine level during hyperoxia in rats

During hyperoxia monoamine oxidase type A acquires the ability to deaminate polyamines and histamine. A preliminary injection of clorgyline (a monoamine oxidase type A inhibitor) before hyperoxic exposure leads to a significant removal of oxygen seizures and prevents changes in the cerebral spermidine and histamine content observed in the unprotected animals. The data confirm the important role of modification of catalytic properties in monoamine oxidase in the mechanism of oxygen intoxication.     

Homocarnosinase activity in the rat brain areas kidneys under different states of hyperbarooxygenation

The homocarnosinase activity in different brain areas and kidneys of the normal rats and under different conditions of hyperbarooxygenation are determined. The highest activity of this enzyme is observed in cerebellum. The high homocarnosinase activity is typical of kidneys as well. The action of oxygen in a dose of 0.425 MPa for 60 min (in the absence of convulsions) increases the homocarnosinase activity in the cerebral hemispheres by 18.6%, in the midbrain by 18.6%, in midbrain and diencephalon--by 56.5%, and in the medulla oblongata--by 40.6%. The homocarnosinase activity in the cerebellum decreases by 16.7%, in kidneys--by 18.5%. At the convulsive stage of oxygen intoxication caused by the effect of 0.7 MPa dose of oxygen the homocarnosinase activity in cerebral hemispheres rises by 158.5%, in the midbrain and diencephalon--by 141.5%, in the medulla oblongata by--161.1%. Under the same conditions homocarnosinase activity in the cerebellum is unchanged as compared with the control.     

Prevention of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity.

Toxicity to the central nervous system (CNS) by hyperbaric oxygen (HBO) presumably relates to increased production of reactive oxygen species. The sites of generation of reactive oxygen species during HBO, however, have not been fully characterized in the brain. We investigated the relationship between regional generation of hydrogen peroxide (H2O2) in the brain in the presence of an irreversible inhibitor of catalase, aminotriazole (ATZ), and protection from CNS O2 toxicity by a monoamine oxidase (MAO) inhibitor, pargyline. At 6 ATA of oxygen, pargyline significantly protected rats from CNS O2 toxicity whereas ATZ enhanced O2 toxicity. In animals pretreated with ATZ, HBO inactivated 21-40% more catalase than air exposure in the six brain regions studied. Because ATZ-mediated inactivation of catalase was H2O2 dependent, the decrease in catalase activity during hyperoxia was proportional to the intracellular production of H2O2. Pargyline, administered 30 min before HBO, inhibited MAO by greater than 90%, prevented ATZ inhibition of catalase activity during HBO, and reversed the augmentation of CNS O2 toxicity by ATZ. These findings indicate that H2O2 generated by MAO during hyperoxia is important to the pathogenesis of CNS O2 toxicity in rats.     

Effect of chlorgyline on gamma-aminobutyric acid levels in hyperoxia

Chlorgyline, an inhibitor of type A monoamine oxidase, administered to rats prevented oxygen-induced decrease in gamma-aminobutyric acid brain content in hyperoxia. At the same time chlorgyline had an overall protective effect, increasing two-fold the period prior to the development of oxygen convulsions. The data suggest an important role of the modification of monoamine oxidase catalytic properties in the development of oxygen-induced intoxication and, particularly, in the decrease of gamma-aminobutyric acid content in hyperoxia.     

Cytochrome C oxidase activity and oxygen tolerance

Most cultured cells and intact animals die under hyperoxic conditions. However, a strain of HeLa cells that proliferates under 80% O(2), termed "HeLa-80," has been derived from wildtype HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. The tolerance of HeLa-80 cells to hyperoxia is not associated with changes in antioxidant defenses or susceptibility to oxidant-mediated killing. Rather, under both 20 and 80% O(2), mitochondrial reactive oxygen species (ROS) production is approximately 2-fold less in HeLa-80 cells, likely related to a significantly higher cytochrome c oxidase (COX) activity ( approximately 2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. We now report that in HeLa-80 cells elevated COX activity is associated with a >2-fold increase in the regulatory subunit COX Vb, whereas expression levels of other subunits are very close to wild type. Small interfering RNA against Vb selectively lowers COX Vb expression in HeLa-80 cells, increases mitochondrial ROS generation, decreases COX activity 60-80%, and diminishes viability under 80% (but not 20%) O(2). In addition, overexpression of subunit Vb increases COX activity and decreases ROS production in wild-type HeLa-20 cells, along with some increase in tolerance to hyperoxia. Overall, our results indicate that it is possible to make cells tolerant of hyperoxia by manipulation of mitochondrial electron transport. These observations may suggest new pharmaceutical strategies to diminish oxygen-mediated cellular damage.     

Homocarnosine content and homocarnosine-carnosine synthetase activity in brain areas of hyperoxic rats

The homocarnosine content and homocarnosine synthetase activity were studied in the brain of rats in normal state and under hyperoxia. The homocarnosine content is higher in phylogenetically old brain areas as compared with that in the cerebral hemispheres. Its nonuniform distribution in the brain is associated with different activity of homocarnosine-carnosine synthetase in the corresponding brain areas. At the preconvulsive stage of oxygen poisoning the homocarnosine content in all the brain areas does not change, the homocarnosine-carnosine synthetase activity is 32% lower. At the convulsive stage of hyperoxia the homocarnosine amount in the cerebral hemisphere decreases by 33%, in the midbrain and diencephalon -- by 70, in the medulla oblongata -- by 60, in the cerebellum -- by 58%. The decrease in the homocarnosine content correlates with that in the activity of homocarnosine-carnosine synthetase in the corresponding brain areas; in the cerebral hemispheres -- by 33%, in the midbrain and diencephalon -- by 50, in the medulla oblongata -- by 49, in the cerebellum -- by 40%.     

Resuscitative Effect of Hyperoxia Fluid on High-Altitude Hemorrhagic Shock in Rats and Antishock Mechanisms

Pathophysiological characteristics of hemorrhagic shock at high altitude are different from that at plain which involve severe injury, high mortality, difficult treatment and compromised liquid tolerance. High-altitude pulmonary/cerebral edema and multiple-organ dysfunction render the conventional treatment ineffective. Herein, we evaluated the resuscitation effects of hyperoxia solution on high-altitude hemorrhagic shock in rats. For this purpose, a rat model of high-altitude (3,658 m) hemorrhagic shock was established on the plateau and hyperoxia solution (4 ml/kg) was infused through external jugular vein for resuscitation at 60 min post-hemorrhage. Blood pressure, blood gas, left and right ventricular pressure, lung and brain water content, survival time, survival rate at 2 h, levels of inflammatory cytokines and free oxygen radicals in blood and tissue were determined. After resuscitation with hyperoxia solution, blood pressure, arterial oxygen partial pressure, left and right ventricular systolic pressure, ±dp/dt max, survival time and rate were significantly increased. Lung and brain water content were unchanged, malondialdehyde activity in lung, brain and plasma and levels of TNF-α, IL-1, IL-6, and endothelin were significantly decreased. Besides, CGRP was elevated with reduced injury and improved lung and kidney functions. Concludingly, resuscitation with hyperoxia solution is feasible and more effective than other classical liquids, making it the first choice of treatment for high-altitude hemorrhagic shock.     

Changes in the oxidation-reduction system of glutathione--glutathione reductase [NAD(P). H] in the blood and tissues of white rats exposed to high and low temperatures

The content of different forms of glutathione and the NADPH-dependent glutathione reductasa activity in blood, brain, liver and sceletal muscle of the white rats exposed to low and high temperatures have been investigated. It was shown that in general cooling of animals the amount of glutathione in blood, brain and liver increases, the glutathione reductasa [NAD(P)H] activity decreases. Under super-heating conditions concentration of all forms of glutathione in blood and tissues and the glutathione reductasa activity reduce. Data obtained permitted to assume the considerable role of the changes of the glutathione-glutathione reductasa system state in the realization of temperature factors action.     

Lung injury and complement activation: Role of neutrophils and xanthine oxidase

Evidence is presented that oxygen products generated from xanthine oxidase (XO) may also be involved in the pathogenesis of neutrophil-mediate lung injury following intravascular activation of complement with cobra venom factor (CVF). CVF injection in rats resulted a rapid increase in plasma of both XO activity (but not xanthine dehydrogenase) and its reaction product, uric acid. These changes were greatly attenuated in allopurinol-treated animals. The apperance of XO activity was paralleled by a raise in plasma of histamine. Prevention of histamine release by pretreatment of rats withy cromolyn abolished both the rise in plasma histamine and the increase in XO activity. Since we have previously shown that histamine can enhance XO activity in vitro and in vivo (Am. J. Pathol. 135:203, 1989), these observations suggest that the increase in plasma XO activity following CVF injection is related to the appearance in plasma of histamine. Accordingly, pretreatment of rats with xanthine oxidase inhibitors (allopurinol, lodoxamine) or prevention of histamine release by pretreatment with cromolyn significantly attenuated development of lung injury following injection of CVF. Our data support the concept that oxygen radicals derived from both neutrophils and XO are playing a role in the CVF-induced acute lung injury.     

Characteristics of the intensity of glycolytic and oxidative processes in rat brain tissue under hypoxia and hyperoxia

Effect of hypoxia and hyperoxia on some indexes of energy metabolism was studied in the brain of intact rats and those with preliminarily administered hydrocortisone. So hypoxia (8 and 5.5% of oxygen in the medium) increases considerably the lactate and pyruvate content and has no effect on the oxidation and photophosphorylation in the brain mitochondria. The hyperoxic medium (1 at. abs. for 3-5h) inhibits consumption of oxygen and inorganic phosphate by the brain mitochondria, does not effect the lactate content and lowers the pyruvate level. The preliminary administration of hydrocortisone (1 mg per 100 g) under conditions of hypoxia and hyperoxia leads to the further intensification of the glycolysis independently of the initial level of the process. At the same time hormone administration favours normalization of the oxidative processes in the brain tissue under conditions of hyperoxia.     

The cytochrome P-450 content and catalytic activity in rat liver microsomes during hyperbaric oxygenation]

The effect of different conditions of hyperbaric oxygenation (HBO) on content and catalytic activity of cytochrome P-450 in rat liver cytochrome has been studied. The intensity of lipid peroxidation in microsomal membranes has been determined by the content of dien conjugates and Schiff's bases. The rate of amidopyrine demethylation has been shown not to change, but the rate of aniline hydroxylation decreases on 34, 57 and 64% under increase of oxygen pressure up to 0.3, 0.5 and 0.7 MPa correspondingly. The level of dien conjugates increase on 66-87% under all studied conditions of HBO. Cytochrome P-450 content decreases on 45% under the action of 0.7 MPa, but content of Schiff's bases increases on 210% as compared with control.     

The effect of hypoxia and of subsequent baro-oxygenation on the function of the microsomal oxidation system in the rat liver]

The increase of cytochrome P-450 by 34% and its catalytic activity with substrate amidopyrine by 57% as compared with control has been shown under hypoxia (0.029 MPa, 1 h). Hyperoxia (0.2 MPa, 1 h) increases the metabolism of amidopyrine by 148%, benzo[a]pyrene by 158% and aniline by 114% and consecutive affection of hypoxia and hyperoxia--by 247, 45 and 138% correspondingly at fixed cytochrome P-450 amount in both series. The amount of diene conjugates and Schiff's bases under hypoxia increases by 40 and 69% correspondingly, the activity of SOD and catalase decreases by 25 and 23%. The activity of hyperoxia raises the diene conjugate content by 19% at all this SOD activity increases by 95%. Consecutive affection of hypoxia and hyperoxia increases the level of diene conjugates and Schiff's bases by 26 and 23% correspondingly, without changing SOD and catalase activity. The relative microsomal viscosity of lipid layer and zones of enzyme-lipid contacts decreased by 20 and 24% under hypoxia, but under hyperoxia and consecutive affection and hypoxia and hyperoxia it increases by 29-28% and 56-40% correspondingly.     

The effect of hyperbaric oxygenation on the antioxidant status of Xenopus laevis after its preliminary adaptation to oxygen]

We studied the level of lipid peroxidation and the activity of antioxidant enzymes (superoxide dismutase and catalase) in various tissues of adult Xenopus laevis after an initial exposure to hyperbaric oxygenation at the developmental stage 38. We have found that irrespective to the mode of treatment, the level of lipid peroxidation and activity of antioxidant enzymes in the brain, lungs, and blood of these animals were higher as compared to control animals. We demonstrate that, after the exposure of adult animals to hyperoxia, if they were earlier subjected to hyperbaric oxygenation (0.2 MPa) at stage 38, there was no intensification of lipid peroxidation or changes in the activity of superoxide dismutase and catalase. In adult animals initially subjected to hyperbaric oxygenation at the same stage of development but at the pressure--0.7 MPa, the second exposure to hyperoxia led to a drastic intensification of lipid peroxidation in the brain; in some animals, an increased level of lipid peroxidation products in the lungs was observed.     

Biochemical indicators of oxygen poisoning]

Content of extra erythrocyte hemoglobin (EEH), total peroxidase activity (TPA), tseruloplasmin oxidase activity (TOA) and chemiluminescence intensity have been investigated in plasma. The dose-dependent changes of EEN, TPA, TOA are shown in experiments on the animals under hyperbaric oxygen (HBO) pressure (0.2, 0.3, 0.5, 0.7 MPa from 30 min. to 120 min.) and EEH, TPA--under hyperbaric air (1.1 MPa; 4 hours). All these parameters have been studied in patients treated with HBO for the assessment of HBO efficacy in the clinical investigations. These biochemical parameters may be used as informative tests of the functional state of the organism under HBO and hyperbaric air.     

The effect of carnosine on indicators of free radical lipid oxidation during acute stress in rats

The effect of carnosine intraperitoneal injection in rats (in doses 0.2, 2.0 or 20 mg/kg) on the vegetative parameters (arterial blood pressure, Hildebrandt index), the content of free radical oxidation (FRO) products and superoxide dismutase activity in serum and brain homogenates and brain lipid composition under normal condition and after different stress forms have been investigated. The carnosine injection in dose 20 mg/kg preserves and increase in arterial pressure and Hildebrandt index at all steps of stress development. The phase non-unidirectional changes in studied biochemical parameters have been revealed depending on the level of stress development in animals under control. The unidirectional and dose-dependent changes of phospholipid content and the level of brain lipids, decrease of FRO products in tissue and brain cholesterol, the increase of the superoxide dismutase activity of serum and brain homogenates have been found in intact and stressed animals after carnosine injection. A comparison of carnosine pharmacokinetics with concentration dependences of the antioxidative effect under in vitro and in vivo experiments comes to conclusion concerning the carnosine indirect adaptogenic action.     

Effect of clorgyline on the intensity of lipid peroxidation and on erythrocyte membrane stability in hyperoxia

Administration of monoamine oxidase type A inhibitor clorgyline to rats before hyperoxia prevented oxygen-induced increase in diene conjugate and Shiff's base brain and plasma levels in hyperoxia. This was due to antioxidative effect of clorgyline which resulted in stabilization of blood cellular membranes. Clorgyline had a normalizing effect on extraerythrocyte hemoglobin level, total peroxidase activity and glucose-6-phosphate dehydrogenase activity in the serum.     

Effects of (S)-alpha -fluoromethylhistidine and metoprine on locomotor activity and brain histamine content in mice.

We examined the effects of (S)-alpha -fluoromethylhistidine (FMH), an inhibitor of histidine decarboxylase, and metoprine, an inhibitor of histamine N-methyltransferase, on the locomotor activity and the brain histamine content of ICR mice. The brain histamine content was decreased by FMH (12.5 or 50 mg/kg, i.p.) and increased by metoprine (4 mg/kg, i.p.). Under these conditions, the locomotor activity and the number of rearing were significantly decreased and increased by FMH and metoprine, respectively. The higher the brain histamine content, the greater the locomotor activity and vice versa. In a previous paper [Sakai et al., Life Sciences, 48, 2397-2404 (1991)], we showed that thioperamide, a histamine H3 antagonist, which enhances the release of histamine from histaminergic neurons, in doses of 12.5 and 25 mg/kg, i.p. increases the locomotor activity, whereas it decreases the brain histamine content. Taken together, these results support the hypothesis that central histaminergic neurons may be involved in the control of state of locomotion and rearing.     

Effect of arginine on the arginase activity and urea content of the brain and liver of rats acclimating to cold

At cold stress (3 days exposition at 2--4 degrees C) the urea formation in rats brain and liver does not become more active, the content of extraerythrocytic hemoglobin and the total peroxydase activity increase in blood serum, the animals sensitivity to the action of hyperbaric oxygenation (HB0) grows. At cold adaptation (45 days at 2--4 degrees C) the urea content in tissues and the activity of arginase in liver increase, the concentration of extraerythrocytic hemoglobin and the total peroxydase activity normalize, animals become more resistant to HB0. Every day administration of arginine during 3-day cold effect makes the brain and liver arginase on 42 and 28% more active, increases the urea content on 26 and 19%, stabilizes the erythrocytic membranes. The animals protected by arginine against cold are more resistant to the action of HB0.     

Protection from hyperbaric oxidant stress by administration of buthionine sulfoximine.

To explore the role of glutathione in protecting rats from hyperbaric hyperoxia, we administered buthionine sulfoximine (BSO) to block gamma-glutamyl cysteine synthase activity and decrease tissue glutathione synthesis. We then exposed these animals and their vehicle-treated matched controls to 100% oxygen at 4 ATA or room air at 1 ATA. After BSO treatment, glutathione concentrations in air-exposed controls decreased 62% in lung, 76% in liver, 28% in brain, and 62% in plasma. Paradoxically, BSO-treated rats were protected from hyperbaric hyperoxia. The BSO-treated animals seized significantly later and had a markedly prolonged time of survival compared with the vehicle-treated controls. We conclude that BSO treatment protects rats from hyperbaric hyperoxia, despite its effects of lowering plasma and tissue glutathione concentrations. This protection may be related to a direct effect of the compound in decreasing free radical-mediated tissue injury, increasing tissue antioxidant defenses, or increasing seizure threshold.