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MILD STIMULATION OF INTERNAL PEROXIDATIVE PROCESSES BY INHALATION OF NEGATIVE AIR IONS. VITAL SUPEROXIDE EMISSION.

M.N.Kondrashova, V.P.Tikhonov*, A.N.Tikhonov#, E.A.Kosenko, I.A.Stavrovskaya, R.E.Kazakof, T.V.Sirota, A.V.Temnov, V.D.Tcvetkov, E.G. Litvinova.

Institute of Theoretical and Experimental Biophysics, Rus. Ac. Sci., Pushchino; AOOT DIOD*, Moscow; Moscow State University#; Russia.

The elucidation of biophysical mechanism of biophoton generation in the organism is one of important aspects of their study.

Our investigations suggested that biophoton emission can be induced by reactive oxygen species (ROS) - superoxide (SO) and hydrogen peroxide (HP). Their generation occurs in living organisms and is increased by external electrical influences on animals and plants.

Negative air ions (NAI) are vitally necessary.

Pioneering investigations of A. Tcijevsky showed that the presence in air of some negative electrical charges, i.e. negative air ions, NAI is necessary for life [1 - 3]. Their content is high in a clear rural air, under movement of natural waters or air - up to 10 000 negative charges /cm3. Their amount falls in polluted air, in closed rooms and air-conditioned buildings, near TV sets and computers - up to 250 - 100 / cm3 and even to zero.

Chronic deficiency of negative air ions leads to decrease of biological resistance of organisms and to maladies. Animal experiments showed that complete deprivation of NAI leads to death.

Tcijevsky invented generator of negative electrical charges. He proposed to use inhalation of NAI for medical treatment and sanitation.The pronounced healing effects of respiration with air enriched by NAI was observed in different countries. However the primary biophysical mechanism of beneficial biological effect of NAI was obscure before our recent investigations. Therefore the advantages of healing with such natural means as NAI in spite of medical drugs were not clear. That is why now medical application of NAI is forced out by numerous artificial drugs which are in some cases harmful.

Reactive oxygen species (ROS) provide beneficial effect of negative air ions .

Negatively charged oxygen, superoxide serves as the main carrier of negative charges in air [ 3 ].

Even before SO discovery Tcijevsky foresow that ionized oxygen provides healing effect of NAI. Participation of SO in NAI effects was claimed by Krueger, and Goldstein on the reason they are abolished by SOD [ 4, 5 ].

It seems that SO presence in AI directly evidences that the primary mechanism of their action involves peroxidative processes activation. However such data were absent before our experiments. Probably there was psychological cause as peroxidative processes were still presumably considered as damaging but not beneficial. In contrast

more recent data show that ROS in concentrations lower than were studied previously are necessary for vital activity [6-9].

For this reason we suggested that mechanism of beneficial biological effect of SO in the flow of NAI is in the activation of peroxidative oxidation in the organism however at less extent than under pathological processes.

This suggestion was proved by our further investigations which are reviewed below.

Appearance of superoxide and hydrogen peroxide in air and in solutions under influence of air ions.

Formerly we detected the presence of ROS in the flow of NAI generated by source which we used, Tcijevsky Lustre, electroeffluvial ionizer, ELION-131M (DIOD, Moscow) [ 10]. NAI were generated by electrical discharge from negatively charged spikes. Many needles placed along hard wires serves as electrode of the ionizer. Feeding the electrode is provided by high voltage generator of square impulses under 50 kV, 1 Hz, 30 msec. The mean value of ion current is 2 m A. The Lustre is powerful source of unipolar only negative ions. Concentration of air ions was measured by aspiration condenser method with an ion counter SAI-TGU-66m (Tartu, Estonia).

The concentration of AI at the working point in our experiments was from 6 x 105 to 1.2 x 10 6 negative charges in cm3. This is ultra-low concentration: 10 -15 -10 - 12 M. These concentrations are effectively used for profilactic and medical treatment.

SO and HP are connected with interconversion shown in scheme: two molecules of SO spontaneously form HP and water:

This spontaneous reaction is slow. In tissues this reaction proceeds rapidly as it is catalysed by superoxide dismutase (SOD).

 

  • SLOW, SPONTANEOUS
    •

    • O2` · + O2` · + 2H+ - - - - - - - -- - - - - - - - - - - - - - - - ® H2O2 + O2

    RAPID, BY SOD

     

    SO is very short-living substance therefore its detection demands free radical trap. We used tiron (Sigma, USA) which was measured by EPR spectra with Brucker 400 MHz spectrometer. This method showed O2` · appearance with the rate 0.5 - 1.0 m M/min [10].

    HP is more stable substance than SO, this can be accumulated in higher concentrations and therefore can be easier detected. HP was measured by luminol fluorescence in the presence of horse raddish peroxidase. Our investigations first show the appearance of HP in solutions treated with air ionizer. The found concentrations were about 1 m M [11] . It is not widely known that HP in micromolar concentrations is inherent to natural air and waters. Its concentration in natural flowing waters is several m M while in rain and thunderstorm rain this rises to tens and hundreds m M. Moreover it was found that the presence of low concentrations of HP is necessary for life of water organisms. Its exhaustion in stagnant ponds leads to pathology and death of fish fry [12 ].

    In these cases HP or iron ions, inducing peroxidative processes are added to water. This initiates ROS formation and this restore vital properties of water in stagnant ponds but does not poison it. Therefore the presence of ROS in water is as necessary for life of water organisms as the presence of NAI in air for earth animals.

    The presence of ROS can explain vital properties of water from natural sources in contrast to so called artificial or «dead» boiled and distilled water.

    Our investigations first show that treatment with air ionizer restors ROS content in distilled laboratory water.

    Superoxide dismutase (SOD) activation by air ions.

    The next step of our investigations was to find out if AI can effect SOD activity. SOD is the first enzyme which meets SO in cells of the organism. We suggested this to be the primary target of air ions. This suggestion was tested by SOD activity measurements in solutions treated with air ions. SOD activity was measured by standard method using generation of SO by xantine oxidase and measurement NBT reduction. SOD eliminates SO and decrease reduction.

    Our investigations first showed that pretreatment of solutions with air ions increase SOD activity. Different preparations of SOD were tested: hemolisates of rat, duck and human erythrocytes and commercial preparation of isolated enzyme[ 11]. In all cases AI pretreatment increased SOD activity. Activation of SOD by NAI evidences that mechanism of their action is connected with peroxidative processes. This connection became more evident when mechanism of this activation was elucidated. Activation is mediated by low, micromolar concentrations of HP. The results show pronounced stimulatory effect of 1 m M HP on SOD activity.

    SOD activity is also regulated by inhalation of NAI in patients [10].

    Inhalation of superoxide with air stimulates ROS formation in mitochondria.

    Generation of HP in mitochondria (MCH) is the most powerful source of its formation in tissues. The next step of our study was investigation if inhalation of NAI by animal influences HP generation in MCH.

    We used standard system for HP generation which elaborated by B. Chance [13]. HP is a product of SO which is formed by coenzyme Q in semiquinone form. This is provided by addition of succinate with antimycin A (ANT). Heart MCH are used because they do no contain catalase which eliminates HP.

    In order to find out how inhalation of NAI influences ROS formation in MCH we kept rats under Lustre for 35 min. After that MCH were isolated from heart and HP formation and respiration in MCH were measured. We have found that HP formation was considerably rised in MCH after AI inhalation under conditions appropriate for this process [14].

    THESE DATA FIRST SHOW THAT INHALATION OF NAI STIMULATES PEROXIDATIVE PROCESSES IN THE ORGANISM.

    It is important that formation of HP does not damage MCH but accelerates their energy functions. In independent experiments was shown that NAI inhalation improves energy functions and structural states of MCH under stress and adrenaline induced excitement [15-20]. This can be explained by our old data showing that peroxide compounds stimulate oxidative phosphorylation [21].

    Thus the phenomenon found evidences that increase of ROS formation in MCH within physiological concentrations STIMULATES MITOCHONDRIAL ENERGY FUNCTIONS BUT DOES NOT DAMAGE THEM.

    This example shows that any increase of ROS should not be a priori considered as a damaging factor. In contrast this can be favourable for functions.

    OUR GENERAL CONCLUSION IS THAT INHALATION OF EXTERNAL ROS

    INCREASES GENERATION OF INTERNAL ROS IN THE ORGANISM.

    Plants as a source of ROS.

    ROS are formed in ANT - insensitive respiration [13]. The portion of ANT - insensitive respiration is small in animals - within several per cents of total respiration. In contrast in plants ANT-insensitive respiration is greater and can reach 90 %. Therefore production of ROS by plants can be considerably greater than by animals.

    We suggested that well-known stimulatory effect of air near trees, in particular in pine

    forest is due to production of ROS by leaves and needles. These ROS are the same NAI which are favourable for health. We decided to test this suggestion in room plants. It should be considered that room in nature as natural water and air exists under electrical fields. In order to approach artificial conditions of plants isolated from earth to natural electrical environment we applied negative electrical charge to roots of plants through soil in flowerpot. We used the generator of electricity used in Lustre [22].

    We really found that different room plants generate NAI under electization. This amount can reach 2 - 5 thousands charges in cm3. Aloe was found to be better source of NAI comparing to several other room plants.

    The generated ions are the products of metabolic processes but not simple conduction of applied electrical field because generation depends on physiological state of plant.

    Points of growth produce NAI emission.

    Production of NAI is considerably greater in intensively growing young plants comparing to older. In spite of young aloe is 5-fold smaller its total generation is 2-fold greater than the old. The cause is that intensively growing tips of leaves are the main generators of NAI.

    The tips are different in young and old plants. They are of yellow green colour and are mild and fluffy and intensively growing in young plant, while in old plant they are of brown colour, covered by hard smooth skin and do not grow. Total amount of intensively growing tips of leaves is greater in smaller young plant than in bigger old.

    We compared generation of NAI by young aloe with even more intensively growing one-week-old grass. In spite the mass of grass is smaller than aloe generation of NAI by grass exceeds generation by aloe. Even small bush of grass in flowerpot generates up to five hundred thousands NAI in cm3 at the 1 m distance.

    Plant generated NAI contain ROS.

    But one more question should be answered: do contain NAI produced by plant SO as NAI produced by ionizer and mitochondria ?

    We put a strip of paper with nitro blue tetrazolium solution on the tip of leaf. During electrization of plant blue staining was appeared. It was SOD sensitive. This evidence SO production.

    Our findings can explain well known beneficial effect of air near trees and grass and its similarity to healing action of natural waters, which is expressed in ancient wisdom:

    «Have a rest near flowing water and near green grass». It was mentioned above that natural flowing water generates air ions and contained SO and HP. We have shown than green grass is also a source of NAI, which contain SO. Bulgarian folk physician Dimkov stated that grass irradiates energy much more intensive than trees. This can be relevant to SO production by plants and to higher amount of growth points in intensively growing grass. Cutted grass in lawn probably irradiates even more ions because it is maintained in a growing state. Apparently these large green vegetable fields around Neuss serve as a powerful source of stimulating negative charges which maintain mental energy of investigators here at the constant high level.

    ROS production is involved into biophoton emission.

    Thus investigations reported have revealed stimulation of internal SO production and rise its efflux into air in animals and plants under action of electric fields. We found SO emission to be more intensive in points of growth. These points are also most intensive sources of mitogenic radiation, which was discovered by A. Gurwitsch [23]. He also showed that oxygen presence is obligatory for mitogenic radiation. This evidences that SO production is related to mitogenic radiation. Mitogenic radiation is the classic example of biophoton emission. Therefore biophoton emission is dependent on SO production. What can be the mechanism of this coupling? According to the view of V. Voeikov SO induces free radical reactions and therefore electron excited states of biomolecules [9, 24]. This provides general stimulating effect of low concentrations of SO.

    Understanding metabolic mechanisms of biophoton generation allows to regulate their emission.

    ASKNOWLEDGEMENT

    The work was supported by RFFI grant of Leading Scientific School (Russia)

    N95-15-97854

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