National Academy of Sciences
7 Hasratian St., Yerevan 375014
Phone (3742) 281772
Fax (3742) 288427
Correspondence should be addressed to: Prof. Ayrapetyan S.N.
Keywords: infrasound, electrical conductivity, water, DNA, optical density.
The effect of mechanical vibration (MV) on distilled water specific electrical conductivity (SEC) and optical density of DNA aquatic solution was investigated. Distilled water (209x10-6S/m) was treated by MV of several frequencies in the range from 3 to 5,000Hz with an intensity of 90dB for 30 minutes. Different values of water SEC changes were determined. Low frequency (from 3 to 100Hz) MV decreased the SEC of distilled water. The greatest decrease of SEC (by 15.7%) under the influence of MV was observed at a frequency of 4Hz. There was no effect at frequencies higher than 100Hz with the same intensity of 90dB.
The treatment of DNA aquatic solutions by MV of 4 and 10Hz frequencies decreased their optical density by 4.2±1.1% and 4.8±1.2% respectively in comparison with control. In cases of treatment by frequencies of 20 and 50 Hz no significant effect was observed.
The mechanism of MV effect on water probably is caused by changes of system structural characteristics. It is confirmed by experiments with DNA solution, where the decrease of optical density (at 260nm) under MV treatment is conditioned with the increase of the probability of hydrogenous bonds formation between the bases. Obtained data allow us to state that cell water medium can serve as a target through which MV may cause the cascade of cell metabolic processes.
Although the large quantity of data exists concerning the effects of physical factors on cell's functional activity, the nature of cell target for those factors is not clear yet (1). Water is the basic component of cell in which the metabolic processes take place. It is considered that insignificant changes of water physical and chemical states may significantly change the functional activity of cells. By this mechanism the influence of electromagnetic fields on physical and chemical properties of water, salines and biosystems can be explained.
If we assume that mechanical vibrations can influence on probability of formation and structure of hydrogen bonds it could be expected that the MV will change the optical density of DNA solution (DNA is in denatured state). The increase of light absorption at 260nm (called a "hyperchromism") is one of the well known methods of determination of DNA denaturation degree (2, 3). The changes of optical density (changes of light absorption level) may take place due to formation or the break of hydrogenous bonds between the nitretic bases of the molecules.
Previously it has been shown that the water electrical conductivity is one of the most sensitive features reflecting the changes of the physical and chemical properties of water (1, 4). The aim of this study is the investigations of frequency-dependent influence of MV on distilled water SEC and optical properties of DNA aquatic solution.
MATERIALS AND METHODS
The experiments were performed on the equipment that allowed to treat water with MV (with frequencies 3 to 5,000 Hz with intensity 90dB) with simultaneous detection of the SEC to an approximation of not more than 0.1%. The block-scheme is shown in figure 1.
The glass vial 1 (diameter 1sm, volume 5ml) with two electrodes inside 2 is firmly connected with the vibrating part of the equipment 3'. The electrodes are made as platinum strips with 1sm2 surface, standing in 0.5sm distance from each other.
The electrodes are connected with electrical conductivity measuring instrument 4, which is able to measure the SEC of water using an alternating current (10-9A, 70 Hz). As the conductivity is measured in micromode regime (5), the use of low noise voltage amplifier of alternating current is increasing the accuracy of measurement by excluding the influence of thermal effects. The increase of accuracy of measurement is also provided by excluding the effect of measured cell capacity and the parasitical capacity of connecting wires, due to the use of the phase-sensitive cascade - a synchronous filter with a detector. The use of such a cascade in the chain of signal processing leads to the increase of the stability from the electric noise (5). For uninterrupted registration of SEC the recorder is connected with the output of the measuring instrument 5.
The vibrator is controlled by the generator of sinusoidal signals 9 (G3-118, Russia) and is vibrating vertically in a certain frequency and intensity. Thus the MV is transferred to the containing of the vials with minimal waste. The amplifier 8 was used to coordinate the generator high output impedance with the vibrator low input impedance. The frequency and intensity of MV were checked by frequency-measuring instrument 10 (Ch3-47A, Russia) and intensity measuring instrument 7 (EVCh-2, Russia), the latter receives signals from sensing element 6 placed on the vibrating part of the vibrator. This scheme of intensity control allowed us to maintain the intensity volume on the same level for all using frequencies (even for resonance frequencies that are higher than 200Hz for the given equipment) and avoid the influence of resonance of both mechanical and hydrodynamic natures.
The non-linearity of the systems for 90 dB intensity was no more than 1%.
All the experiments were performed in conditioned rooms, with a mean temperature of +20±0.5oC. The atmosphere pressure was 87.8±0.5kPa.
The SEC was recorded in 5ml volume of distilled water with or without the influence of MV of certain frequency for 30 min. The SEC values were recorded for that period and after 10 experiments the mean value, standard deviation and P value were calculated (Student's t-test) for every certain frequency.
Parts 3, 6, 7, 8 and 9 of described equipment were used for DNA aquatic solution treatment. The vials of 10mm in diameter and 50mm in height were used. The optical density of DNA solution was measured by spectrophotometer (SPh-46, Russia) at 260nm.
To avoid the solutions aeration while treating, the vials were closed in such a way that the air quantity in the vials was insignificant (the vial volume is approximately equal to vial containing solutions volume).
The DNA from calf thymus (Serva, USA) was used (2.5x10-5M). The concentration was determined by absorption method (extinction coefficient at 260nm - 6400M-1sm-1) The aquatic solution of DNA in 3ml was treated by MV with the intensity of 90dB for 30min. Then the optical density of solutions was measured. The control solution was prepared in the same way and left without treatment for the same time untill measuring. Ten experiments were carried out for each probe.
For elucidation the frequency-dependence of MV influence on distilled water SEC the following frequencies were used: 3; 4; 5; 6; 10; 15; 20; 25; 50; 100; 150; 200; 250; 500; 1,000; 2,500 and 5,000Hz. As it is shown in figure 2, the maximum decrease of water SEC by 15.7±2% (p<0.1) was observed at the frequency of 4Hz. Increasing the frequency of MV the less value of water SEC decrement was observed (see figure 2).
For elucidation MV influence on probability of formation hydrogen bonds between base-pairs, in the next series of experiments the MV influence on DNA solution optical density (at 260nm) was studied. The frequencies were chosen according to data of previous experiments - 4; 10; 20 and 50Hz, some frequencies were from the "window" where the effect was observed. Frequencies of 4 and 10Hz decreased the optical density by 4.2±1.1% and 4.8±1.2% respectively (see figure 3), while not significant changes in optical density were observed with 20 and 50Hz frequencies of MV.
DISCUSSION AND CONCLUSIONS
The treatment of distilled water by MV leads to the frequency-dependent changes of its SEC. The present data show that there was a frequency-dependent effect of MV both on distilled water SEC and DNA aquatic solution optical density. Moreover, in both cases the treatment of water medium by frequencies of MV less than 10Hz was more effective. It could be likely connected with the dipole moment of water molecule but the problem needs a more comprehensive investigation.
It's obvious that preparation of solutions is accompanied by mechanical influences. Moreover, in the major part of biological experiments the investigated objects are often perfused with soline which itself is a treatment. Thus perfusion itself can change the physical and chemical properties of water solution and those mechanical influences can lead to the effect on the investigated biosystems. However from this point of view the problem has not been examined yet.
In literature there are data on some temporary changes of water properties after weak mechanical influences and displacements. Thus the temporary changes of SEC were recorded during the bidistilled water (128.7x10-6S/m) was displaced. However the effect was not observed when water was under the metallic screen (6). Probably, the influence has an electromagnetic nature as the metallic screen removes the effect. Besides, such kinds of experiments do not provide the identification of MV by frequency. The equipment (see figure 1) used in present investigation was worked out by our group and allowed us to elucidate the frequency-dependent effect of MV.
The obtained data have shown that low-frequency and infrasound MV decrease SEC of distilled water and optical density of DNA solution (at 260nm). Within the used frequencies the greatest effect was observed at frequency of 4Hz. It is possible that the frequency "window" can be shifted depending on temperature, atmospheric pressure changes and the concentration and the type of dissolved substances (6).
The mechanism of MV actions on water can be correlated with changes of structural characteristics of the system. The data of experiments with DNA solution, where the decrease of optical density was observed under the influence of MV, can be explained by the increase of hydrogenous bonds (between bases) formation probability. The present results allowed us to suggest that the probability of the hydrogen bonds formation can serve as a target for biological action of MV (4). Further more detailed investigations are required for the final conclusion on the mechanism of MV influence.