Effect of Uro-Biophon Apparatus Radiation on Barrier Properties of Bacterial Cell Plasma Membranes

V.M.Fomchenkov, N.B.Borisov, A.N.Bydus, S.I.Petrenko

Obolensk, Moscow Region, 142279

The urogenital URO-Biophon Apparatus for IR-therapy (further in the text referred to as “the apparatus”) developed on the basis of the Russian Federation patent and manufactured by the state enterprise “Izhevsk Mechanical plant (Izhevsk), spec: 944-001-0196638-98 has proved its effectiveness in treating a number of infectious diseases and it has been authorized by Ministry of Public Health for its applying in clinics. However, the knowledge of the apparatus radiation mechanisms effecting various functional systems of microorganisms cells is not sufficient yet. It is required to perform a series of research works for better understanding basic functions damaged by radiation that will lead to death of microorganism cells. The change in the barrier function of plasma membranes is one of the cell damage indicators (1). The barrier properties of bacteria cell plasma membrane (PM) is of considerable importance in maintaining their energetic status and cell homeostasis as well as in transporting nutrients and secreting metabolism products. The variation of the membrane barrier function was estimated by a number of indicators using the following methods:

- Spectrophotometry on the basis of estimating leak out of the UV absorbants.

-Electroorientational spectroscopy using the “Oreol” unit.

- Method of cell electrophoresis.

- Absorption of substrates labeled with a radioisotope.

Materials and Methods of Investigation

The object of the study were the following bacteria: Escheria Coli K-12 and Chlamydia trachomatis. E-coli was cultivated in the flask with an enriched glucosopeptone medium at 37⁰C for 4 hours, logarithmic growth phase, and 12-14 hours, stationary phase of culture growth. Ch. trachomatis was cultivated for 72 hours in yolk sacs (viteline sac) using 7-day chicken embryo.

The viteline sacs were suspended in a weak buffered physiological solution and clarified by centrifugation at 2500-300g. A decrease in number of E coli viable cells was determined by comparing the number of colony forming units grown for 24 hour period in the solid medium for control and irradiated specimens. A decrease in number of Ch. trachomatis viable cells was estimated by comparison of minimal cultivations responsible for embryo infection. UV absorbant leak out was determined by measurement of optical density of a supernatant received by spinning down cells of microorganisms at 25000g and 26nm in 1cm – flask using the DV-7 spectrophotometer produced by Beckman. The supenatant of unexposed cells served as a basis for control. The electroorientational spectroscopy of cells consists in analyzing their electroorientational effect-frequency relationship in the range of electric field with frequency variation from tens Hz to tens MHz. The electroorientational effect (EOE) is defined by variation of optical density of variable electric field that is initiated by lining up cells with their axes along or across the electric lines of force. The experimental plant is similar to the described before (z). The electrosurface properties of cells were analyzed by laser Doppler Spectroscopy using Zetasizer-2 apparatus produced by Malvern, England.

The transport processes were investigated with application of 14C substrates by the procedure described in book (3), V-14C glucose (Specific activity 1.0mku/mmol and 14C glutamic acid (4mku/mmol) were used for this work. The radioactivity was defined by the “Ultrabeta 1210ËÊÁ scintillation counter.

Results and Discussion

The apparatus effect on microorganism cells was investigated by a single and multiple irradiations (5-times) of the cell suspension in the nutrient medium. Unexposed cells served as a standard (a control specimen). For avoiding the undesirable apparatus effects on control cells, the radiation exposure was performed in the room at a distance of 10 meters from the disposition of the control specimen where further works were accomplished. The results of comparative study of changing viability of the E coli cells irradiated by the apparatus are given in Table I.

Table I

One can see from the results given in the table that the number of viable E coli cells has substantially decreased after processing by the Biophon apparatus. It is notable that repeated irradiations do not lead to further decreasing in viability of the cells. In the apparatus effecting on ch. trachomatis, the increase of minimal infection dose by an order was observed, compared to the control specimen.

It should be emphasized that we have not received an increase in the degree of exposure when increased the radiation exposure number. On treatment of the cells suspension, the aliquot in volume of 3ml was centrifuged using an ultracentrifuge and then the absorption was defined at 260nm in the supernatant, in this case the unexposed supernatant served as controls. If the supernatant absorption was more than an optical unit then the supernatant was so diluted with distilled water that the optical density could lie in the range of 0.3 to 0.8. It has been found that the irradiation will initiate statistically significant increment in optical density of the supernatant by 20-30% for both microorganisms and again we have certified the independence of the value on irradiation number.

In recent years it has been shown that the elctrophisical methods, specifically, the electroorientational spectroscopy for analyzing bacterium cells damage (4) are very promising. In this case the main indicator of cell damage is modification of electroorientation spectrum. The value of electroorientational effect (EOE) at low frequencies (less than 10kHz) is defined by the electrosurface properties of the cells as well as conditions of the double electric layer on the boundary “cell-environment”. At frequencies above 10kHz, the EOE value is defined by the barrier properties of cell membranes as well as by the concentration and mobility of cytoplasma ions. Besides, over the whole range of frequencies the EOE value of the cells suspended in medium with certain conductance and pH depends on the cell size and form, as well as on their refraction index. The frequency dependence of the cell EOE is referred to as electroorientational Spectrum (further referred to as EO) and it will characterize the cell surface condition (at low frequencies) and intracellular contents (at high frequencies). As a result, by comparison of EO-spectra of intact cells and cells exposed to the apparatus radiation we can evaluate its action on the cell surface structure as well as its effect on the barrier properties of plasma membranes. It should be kept in mind that because of distinctions between EO spectra of the intact cells of various kinds and the pecularities of their alternations as a result of cell damage, the optimum choice of frequencies in the range of KHz and MHz for defining modifications of the high frequency region of EO-spectrum characterized by b value (preceding reference), may be different depending on the cell kind and a damaging factor. The relative decrease in b -value for the cells exposed to the apparatus radiation studied against the controls (negative values D b /b ) points to the fact that the barrier of PM cells permeability was damaged.

Fig.1-4 present EO spectra of the E coil cells and Fig.5-6 EO spectra of Ch. trachomatis, intact and irradiated; the spectra were acquired immediately on irradiation (Fig.1, 2, 4) and six hours after exposure to radiation (Fig.3, 4 and 6). For E coli we acquired spectra of energized (with 0.2% glucose) cells (Fig.1 and 3) and not energized cells (Fig.2 and 4). In this case D b /b values were calculated (by values defined from EOE magnitude at frequencies 0.1 and (0mHz); the values of D b /b are given in Table 2.

Table 2.

The data presented above show that the cell irradiation by the apparatus is reduced to two basic effects. In the first place, the electrosurface properties of cells may greatly vary at low frequencies, as a rule, the low frequency region of EO Spectrum of the irradiated cells runs substantially below that of the intact cells. The exception was EO Spectrum of energized cells within six hours after irradiation (Fig.3), when the low frequency region of the spectrum lay well above in comparison with the intact cells.

In the second place, in the range of high frequencies we observed, more or less pronounced modifications typical for damaging barrier properties of the bacterial cell plasma membranes (negative values of D b /b ). This effect is more distinct for energized cells immediately after irradiation, then it will increase in the course of time (within six hours after radiation). The values of electrokinetic potential for intact and radiated cells determined in the buffer solution of phosphate and citrate with pH 7.0 and 2.6 at ionic force equal to 0.02 are given in Table 3.

Table 3.

From Table 3 it is evident that the cells exposed to radiation could not give rise to significant variation of electrokinetic potential except for electrokinetic potential of the energized cells at pH2.6 within five hours after their radiation. The latter correlates with anomalous variation of the low frequency EO spectrum region of the cells (Fig.3). These peculiarities are likely to be directly related to the substance leak out of the cells and their adsorption on the cell surface. The possibility of that is validated by high negative D b /b values of these cells (Table 2). Thus, EO Spectroscopy also has revealed pronounced variation of the electrosurface properties of the cells irradiated by the apparatus.

Changing EO spectrum of the irradiated cells in the range of high frequency spectrum roll off indicates small but noticeable violation in the barrier properties of cell plasma membranes (compared to resisting cells) and its magnitude will increase in the course of time after the cell radiation. The information on cell electrophoresis testifies that the electrokinetic potential of the cells on radiation by the apparatus changes moderately. We have made an attempt to evaluate effect of changing PM barrier properties on the rate of nutrient absorption from medium. For this purpose, the cells were spinned down and suspended in 0.1 M phosphate buffer. 0.2% - glucose or glutaminic acid with an additive of radioisotope labeled analogs were added to the suspension. On incubating for 30 minutes, the aliquots were selected and applied on 0.22m filter. The filter was washed with a buffer solution for removing radioactive impurities from the medium, than the cell radioactivity was determined by the liquid-sample scintillation counter. Thereafter, the filter was further washed with 10% cold trichloroacetic acid and then the cell radioactivity was determined once again. The difference of two estimates were taken as the substrate amount included in polymer components of the cell. The results are given in Table 4. Absorption of radioisotope labeled substrates by the intact and irradiated E coli cells.

As the given results indicate, the irradiated cells are slightly more active in absorbing substrates from the medium, it is particularly evident for glucose. However, the rate of including a label from the substrate to the polymer components drops steeply. It is likely to be associated with decreasing rates of biosynthetic processes in the irradiated cells.

In summery, we can draw the following sufficiently justified conclusions:

The “Biophone” apparatus radiation will result in variation of plasma membrane permeability; as a result of this processing, the sublethal damage of cells will be caused and their future will depend on their ability of restoring damaged systems (or inability of restoring).

Literature:

1. N.N.Nickolsky. In book: Common mechanisms of cell reactions on damaging exposures. Edition 17.L.,1977,p.23.

2. A.Y.Ivanov, V.M.Fomchenkov, F.F.Miroshnikov. Electronic material processing, 1984 No4,p.53.

3. L.A.Osterman. Investigation of Biological Micromolecules by Electric Focusing, Immunoelectrophores is and Radioisotopic Methods. M.Science, 1983, p.213.

4. A.I.Miroshnikov, V.M.Fomchenkov, A.Y.Ivanov. Electrophysical Analysis and Separation of Cells. M.Science, 1986,p.184.