M. N. Kondrashova

Preservation of the in vivo state of mitochondrial network for ex vivo physiological study of mitochondria

Over the course of many years our laboratory has been engaged in the study of physiological functions of mitochondria ex vivo. We showed that the unavoidable destruction of mitochondrial-reticular network during traditional isolation of the mitochondria diminishes the observable ex vivo changes of mitochondrial processes in vivo. Comparing preparations obtained from quiescent and stressed rats, we found that the great difference in size of assemblies of mitochondria preserved in homogenate disappears when it is diluted for the measurement of respiration. This also leads to a decrease in the difference between respiration of mitochondria from quiescent and stressed animals. We developed a new method that provides ex vivo stable preservation of the in vivo network using a cytochemical procedure on glass-adhered lymphocytes in blood smear. We radically changed the incubation medium for the measurement of dehydrogenase activity that excludes an artefact of succinate dehydrogenase hyperactivation ex vivo by non-physiological components of the traditionally used solution. Our method made it possible to observe ex vivo two- to eightfold increase in succinate dehydrogenase activity by adrenaline in vivo, while the activity of alpha-ketoglutarate dehydrogenase changed reciprocally. The data obtained show that the structure changes of the network play an important role in physiological regulation of mitochondrial functions. Thus, it may be possible to correct mitochondrial dysfunctions in the organism by substances supporting the stability of mitochondrial network. The developed method is non-invasive, informative and, therefore, is convenient for clinical investigations, particularly of mitochondrial diseases.

Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria.

Conditions for the realization in rats of moderate physiological stress (PHS) (30-120 min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity. These mitochondrial markers undergo an initial 10-20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2-3-fold by 120 min of PHS. By 30-60 min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120 min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST. The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode. The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy.

The Effect of Inhaled Air Ions Generated by Technical Ionizers and a Bioionizer on Rat Trachea Mucosa and the Phagocytic Activity of Blood Cells

It was shown for the first time that inhaling negative air ions (NAI) leads to changes in the functional state of the rat trachea mucosa, which in turn affects the phagocytic activity of blood cells. The effect depends on the concentration of the NAI, the duration of their action, and the sources of the NAI generation. For air ionization, technical ionizers and a bioionizer (a plant electrified through soil) developed by the Diod Company, Moscow, Russia were used. It was shown that the NAI at high doses (600 000 ion/cm3), generated by an Elion 131M device, induce a damage to the rat trachea mucosa, an inhibition of the activity of its antioxidant enzymes, and a decrease in the phagocytic activity of whole blood cells. Low doses of the NAI from the same source (25 000-50 000 ion/cm3) and high doses of the NAI from devices Elion 132S and 132R (320 000-500 000 ion/cm3), and a bioionizer (500 000 ion/cm3) produced a milder effect, activating the secretion of goblet cells of the trachea mucosa without its damage. A possible mechanism of the effect of inhaled NAI as exogenous reactive oxygen species (ROS) on internal processes in the organism is discussed. The involvement of the ROS in the action of ionized air found in the study is probably the general property of different electrical influences on biological objects. Therefore, the result of the investigation may be of interest for a wide circle of specialists dealing with ElectroMed influences

Complex therapeutical effect of ionized air: stimulation of the immune system and decrease in excessive serotonin. H/sub 2/O/sub 2/ as a link between the two counterparts

We investigated the therapeutic effect of breathing ionized air in patients with chronic inflammation and decreased immune reactivity. Air ions were generated by an electroionizer, Tchijevsky Lustre. The air at the place of breathing contained 120 000 negative charges/cm/sup 3/. By chemical nature, the negative air ions are superoxide at a concentration of less than 1 /spl mu/M, and hydrogen peroxide at a concentration of about 1 /spl mu/M. A three-week inhalation, for 1h daily, resulted in a decrease in the level of pro-inflammatory cytokines, the tumor necrosis factor (TNF), and interleukin-1 (IL-1), as well as excessive serotonin, while the level of the anti-inflammatory cytokine interferon tended to increase. These changes in inflammatory mediators are accompanied by a diminishing of the clinical manifestations of inflammation. The finding that NAI decrease the level of pro-inflammatory cytokines TNF and IL-1, and excessive serotonin explains the mechanism of their known curative effect in treatment of burns and opens a new area of their clinical applications for the treatment of other diseases related to the rise of TNF level, such as sepsis and even heart failure. The observed effects of negative air ions can be attributed to their ability to maintain a continuous flow of low concentration of H/sub 2/O/sub 2/, resulting from superoxide and serotonin oxidation. H/sub 2/O/sub 2/ at a low concentration, is a necessary activator of the immune system.

States of succinate dehydrogenase in the organism: Dormant vs. hyperactive (pushed out of equilibrium)

Using an original cytobiochemical method to study oxidation in mitochondria, preserving their native network organization within cells in a blood smear, we have revealed a hyperactive state of succinate dehydrogenase that arises in the organism under physiological stress. This is generally consistent with the notion of non-equilibrium state of enzymes during their activity. The mechanism moderating the succinate dehydrogenase hyperactivity is based on full-fledged functioning of α-ketoglutarate dehydrogenase, sup-ported by oxidation of isocitrate.

Substrate-specific reduction of tetrazolium salts by isolated mitochondria, tissues, and leukocytes

Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate–NBT was reduced more readily during succinate oxidation, while MTT–during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors–malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.

[Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

The biological effects of near infrared radiation (850 nm) modulated by an acoustic frequency of 101 Hz were studied. The study was conducted on rats; the effect was registered by succinate dehydrogenase activity in lymphocytes in blood smears after the administration of an activating dose of adrenaline, which simulates the state of the organism at early stages of a pathogenic action (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals that were activated by adrenaline was shown. Infrared radiation has a normalizing effect via the reduction of the degree of inhibition or activation of the enzyme induced by adrenaline and has no effect on the control animals. Thus, by modulation of the activity of succinate dehydrogenase, infrared radiation regulates energy production in mitochondria that is provided by the most potent oxidation substrate, viz., succinic acid; the effect is especially pronounced under stress.

Changes in lymphocytes induced by oxidation substrates

Lymphocytes in blood smears of children diagnosed with cancer have been investigated and compared to those of healthy children (matching controls). The conventional hematological procedure for smear staining was used to assess the blood formula and lymphocyte indices, and a new staining method involving incubation of cells in a biochemical medium for 1 h was used to assess the activity of succinate dehydrogenase, lactate dehydrogenase, and the ratio of the activities of these enzymes characterizing the interaction of respiration and glycolysis. A dramatic increase of the size of lymphocytes obtained from cancer patients was detected when lactic acid was added to the incubation medium for the biochemical reaction. This effect was neither observed when succinic acid was added nor when blood smears from healthy children of the control group were tested. Structural changes in the patients’ cells were associated with changes in the activity of the enzymes under investigation. The cytobiochemical test that was developed allows increasing diagnostic sensitivity upon assessment of the patients’ state and can contribute to more efficient treatment of malignant tumors.

The effect of cell-phone radiation on rabbits: Lymphocyte enzyme-activity data

The effect of a GSM 900/1800 mobile phone, which is a widespread source of electromagnetic radiation of the microwave frequency in the environment, on rabbits was studied at power densities of 5–7 μW/cm2. The biological effect was recorded by a sensitive method for the detection of the physiological regulation of enzyme activity inside lymphocytes in blood smears. Succinate dehydrogenase, which is the most powerful energy-supply enzyme in mitochondria, and lactate dehydrogenase, which is an enzyme of glycolysis, were measured. The lactate dehydrogenase to succinate dehydrogenase activity ratio was also calculated as an analog of the Warburg effect, which demonstrates the relationship between glycolysis and respiration. After 60 min of mobile-phone exposure each day for 11 days at a moderate dose, the emitted radiation induced a threefold increase in succinate dehydrogenase activity and a twofold decrease in lactate dehydrogenase activity. As a result, the lactate dehydrogenase/succinate dehydrogenase activity ratio falls from 15 to 5, thus indicating that respiration is predominant over glycolysis. The changes develop already after the first exposure and reach a maximum in 4 days. The predominance of respiration is usually considered as a beneficial state of an organism. However, continuous activation of respiration by mobile phone exposure may cause damage to the normal restorative processes that are supported by glycolysis during periods of rest.