Extremely High Frequency Electromagnetic Radiation Enforces ...

Cell Biochem Biophys (2008) 51:97–103 DOI 10.1007/s12013-008-9020-9

ORIGINAL PAPER

Extremely High Frequency Electromagnetic Radiation Enforces Bacterial Effects of Inhibitors and Antibiotics Hasmik Tadevosyan Æ Vitaly Kalantaryan Æ Armen Trchounian

Published online: 17 July 2008 Ó Humana Press 2008

Abstract The coherent electromagnetic radiation (EMR) of the frequency of 51.8 and 53 GHz with low intensity (the power flux density of 0.06 mW/cm2) affected the growth of Escherichia coli K12(k) under fermentation conditions: the lowering of the growth specific rate was considerably (*2-fold) increased with exposure duration of 30–60 min; a significant decrease in the number of viable cells was also shown. Moreover, the enforced effects of the N,N0 -dicyclohexylcarbodiimide (DCCD), inhibitor of H+-transporting F0F1-ATPase, on energy-dependent H+ efflux by whole cells and of antibiotics like tetracycline and chloramphenicol on the following bacterial growth and survival were also determined after radiation. In addition, the lowering in DCCD-inhibited ATPase activity of membrane vesicles from exposed cells was defined. The results confirmed the input of membranous changes in bacterial action of low intensity extremely high frequency EMR, when the F0F1-ATPase is probably playing a key role. The radiation of bacteria might lead to changed metabolic pathways and to antibiotic resistance. It may also give bacteria with a specific role in biosphere. Keywords Extremely high frequency electromagnetic radiation Bacterial growth and survival F0F1-ATPase, N,N0 -dicyclohexylcarbodiimide Antibiotics Tetracycline and chloramphenicol H. Tadevosyan A. Trchounian (&) Biology Faculty, Department of Biophysics, Yerevan State University, 1 Alex Manoukian Str., 0025 Yerevan, Armenia e-mail: [email protected] V. Kalantaryan Radiophysics Faculty, Department of High Frequency Radiophysics and Telecommunication, Yerevan State University, 1 Alex Manoukian Str., 0025 Yerevan, Armenia

Introduction Extremely high frequencies (of 30–300 GHz) electromagnetic radiation (EMR), or millimeter waves, is of significance because of different reasons. The EMR, including those with low-energetic intensity and those with nonthermal action, is widely used in satellite communication, radiometry, radar and remote sensing technology and therapeutic practice (for reviews, see [1–3]). Besides, loworbital systems of cosmic communication and different elements of mobile telecommunication, and in addition, therapeutic devices radiate EMR of mentioned frequency, small and very small doses (the power flux density of 0.005 mW/cm2) of which affect living cells and organisms, including bacteria. Moreover, this proves to be of interest as bacteria and other cells can interact with each other through EMR (for review, see [3]). In such respect, it was found that bacteria, for instance, possess the ultrasonic radiation [4] or reemission of secondary photons in submillimeter frequency range [5]. It has been shown that the coherent (in time) and ‘‘noise’’ (with broadband frequencies and accidentally changing phases) extremely high frequency EMR with low intensity causes different, including bactericidal effects on Escherichia coli and the other bacteria depending on EMR frequency, intensity, polarization and modulation, exposure duration, post-exposure time, mediated and repeated radiation [5–15], for reviews, see [2, 16]. Bacterial growth phase and anaerobic or aerobic conditions, composition of growth media, cell density, cell-to-cell interaction, genetic features, peculiarities of metabolism and membrane properties in bacterial species and strains and others should be also mentioned. Interestingly, the elevated decrease in E. coli growth rate under anaerobic conditions was shown with coherent extremely high frequency EMR (of 45–53

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GHz) [15]. The latest results depend on medium composition and pH, suggesting a role of water in the bacterial effects of this EMR. The basic mechanisms in bacterial and other effects of extremely high frequency EMR with low intensity are little known. Among cellular and molecular mechanisms in bacterial effects of this EMR, different targets are considered: (1) the bacterial membrane, since the changes connected with surface characteristics of plasma membrane, ion and other substances transport across the membrane and energy-conversing processes have been determined [7, 8, 10, 12]; (2) water (H2O), since the changes in cluster structure and properties of H2O molecules leading, for instance, to increase in chemical activity of water or hydration of proteins and other cellular structures have been defined, and these effects might be longterm [1, 2, 15, 17]; (3) the bacterial genome, since conformational changes in DNA and transition of bacterial pro-phages from lysogenic to lytic state appear [16, 18]. However, experimental results are not enough, and moreover, bacterial response to low intensity extremely high frequency EMR should be formed and integrated to understand these mechanisms well. This environmental factor might probably affect cell membrane, its transport properties and enzymatic activity as well as bacterial sensitivity to inhibitors and antibiotics, enforcing, for instance, antibiotic resistance. Here, the results with effects of coherent EMR with the frequency of 51.8 and 53 GHz on E. coli K12(k) growth and their sensitivity to inhibitors and antibiotics are presented.

Materials and Methods Bacteria and Reagents, Bacterial Growth and Assays Escherichia coli wild-type strain K-12(k) was used throughout. The methods for bacterial growth in peptone medium (0.2% peptone, 0.5% NaCl and 0.2% K2HPO4, pH 7.5) in anaerobic conditions upon fermentation of glucose (0.2%) [10, 14, 15, 19] and determination of bacterial specific growth rate, growth lag-phase duration and of bacterial survival in the minimal salt medium (46 mM K2HPO4, 23 mM KH2PO4, 0.4 mM MgSO4, 8 mM (NH4)2SO4, 8 lM FeSO4, pH 7.5 [19] during 4 days [20] were described elsewhere. Bacteria were grown till the stationary growth phase (during 18–22 h). Growth rate was calculated over the interval, where the logarithm of absorbance of the culture at 600 nm increased linearly with time and expressed as 0.693/doubling time, and bacterial survival was determined

Cell Biochem Biophys (2008) 51:97–103

by counting colony-forming units grown on plates with solid medium with glucose when appropriately diluted bacterial suspension samples taken in the same time for every day during surviving in the medium mentioned were plated. The methods applied to prepare whole cells for assays and to isolate right-side-out membrane vesicles [19, 21, 22] as well as to determine energy-dependent proton efflux [14, 19, 23, 24] from whole cells and ATPase activity of membrane vesicles [20, 23], respectively, did not differ from described elsewhere. H+ efflux was determined in the assay medium (100 mM Tris-phosphate buffer, pH 7.5, 0.4 mM MgSO4, 1 mM KCl and 1 mM NaCl [19] upon adding glucose (20 mM) and expressed as the change in H+ activity in mMol/min/1012 cells. Small changes in H+ activity were defined using a selective electrode (type HJ1131B, Hanna Instruments, Portugal) and recorded using a fine potentiometer; electrode readings were calibrated by titration with 0.01 N HCl. Note that without adding glucose changes in H+ activity by bacteria were insignificant. ATPase assays were done by determination of liberation of inorganic phosphate (Pi) in the other medium (50 mM Tris–HCl buffer, pH 7.5, 0.4 mM MgSO4 [22, 25]) with or without 100 mM KCl added. The reaction was initiated by adding 3 mM ATP (Tris salt) to vesicles and terminated at a given time by addition of 5% trichloroacetic acid or 0.1% sodium dodecyl sulphate. Pi was defined spectrophotometrically, and ATPase activity was expressed in nmol Pi/min per lg protein. Bacterial concentration was estimated by an absorbance at 600 nm and a calibrating curve relating the absorbance to the colony-forming units grown on solid media. The protein was determined by the method of Lowry et al. [26] using bovine serum albumin as a standard. When used, bacteria or membrane vesicles were treated with N,N0 -dicyclohexylcarbodiimide (DCCD), 0.05 mM, for 10-min prior assays. DCCD-inhibited value is a difference between the values in the presence and absence of DCCD in parallel measurements. Note that during treatment with DCCD in concentration mentioned and upon transfer from distilled water into the assay mixture, bacterial concentration was not changed. Moreover, during assays, there was no change in bacterial concentration as well. Bacteria were grown and survived at 37°C as well as all assays were done in temperature-controlled chamber at 37°C. Electromagnetic Radiation of Bacteria The radiation of E coli was produced by means of EMR generator, based on backward-wave oscillator G4-141 (with conical antenna radiating the coherent in time nonpolarized electromagnetic waves with frequency within the


The data processed to be statistical from three replicates at least with determination of the standard error and of the Student’s validity criteria (p) [22, 23]; when not mentioned, P \ 0.01 for the differences between the values without (control) and after exposure to EMR.

Specific growth rate (h-1)

0.9 0.8 60 min

0.6 0.5 0.4 0.3 0.2 0.1

Inhibitory Effect of EMR on Bacteria and Sensitivity to DCCD The radiation of E. coli K12(k) by EMR at the frequency of 51.8 or 53.0 GHz led to a significant decrease in the

Control

51.8 GHz

53GHz

Fig. 1 The E. coli K12(k) specific growth rate changes after radiation by EMR at the frequency of 51.8 and 53 GHz. Bacteria were grown in peptone medium with 0.2% glucose at pH 7.5 under anaerobic conditions [19] after direct radiation for 30 or 60 min. In control, cells were without radiation. Average data of three measurements with standard errors are represented

12 10

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+ Tet 51.8 GHz + Tet + Cmp

6

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4 2 0

Results

30 min

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0

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specific rate of bacterial growth (the values for the growth rate in appropriate controls were close to each other and the small difference was not valid; P \ 0.05 for the decrease in the growth rate for the exposure of 30 min at the EMR frequency of 51.8 GHz); the inhibitory effect was reinforced in *2-fold with time of the exposure of 30–60 min and with the frequency of 51.8–53 GHz (for the 30-min irradiation) (Fig. 1). Moreover, EMR radiation changed the following survival of bacteria (Fig. 2). Such results comply with those that the frequency of 51.8 GHz is resonant for E. coli as determined by Belyaev and co-workers ([18] for review, see [16]) and, on the other side, the exposition of 60 min is effective enough to inhibit bacterial growth as

log10 (bacteria.ml )

range of 45–53 GHz, wavelength of 5.6–6.7 mm, correspondingly [14, 15]. The generator assembled in the Institute of Radiophysics and Electronics of the National Academy of Sciences of Armenia (Ashtarak City) did not differ from well-known Russian made generators (for review, see [2]). Bacterial suspension (grown cells concentrated by centrifugation in bi-distilled water to maximal titer and transferred into a plate with a suspension thickness of *1 mm) was affected by the EMI generator in the option of amplitude modulation with a frequency of 1 Hz (frequency stability was 0.05%); the distance from the antenna to the bacterial suspension was *20 cm; for this distance, the power flux density measured using a power meter was 0.06 mW/cm2 (power was equally-distributed in the exposed sample and power reflected to the waveguide system was insignificant). For the others, the conditions of the radiation of bacteria were similar to those described previously [10, 14, 15]. After direct exposure of bacterial suspension, cells were immediately transferred into the fresh growth (with glucose, 0.2%) or assay medium; otherwise, membrane vesicles were isolated from the exposed cells; there is no change in bacterial concentration during exposure. In control, bacteria were subjected to appropriate growth and assays but without exposure to EMR. It should be noted that the effects of this EMR are the same for different concentrations of exposed E. coli cells [9], but they might differ due to the medium composition [12] Moreover, EMR with a power flux density of 0.06 mW cm-2 was found to have no marked effect on the temperature of bacterial suspension during exposure, which was measured using sensitive technique detecting change of 0.1°C. This is in accordance with the results concerning non-thermal effects of extremely high frequency EMI with the power flux density upto some mW cm-2; heating of objects is, as a rule, below 0.1°C (for review, see [1]).

99

1

2

3

4

Time (days)

Fig. 2 E. coli K12(k) survival after radiation by EMR at the frequency of 51.8 GHz and in the presence of different antibiotics. Bacteria grown till the stationary growth phase (18–22 h) were radiated for 60 min, and 4 lM tetracycline (Tet) or 4 lM chloramphenicol (Cmp) was added. For the others, see Materials and methods and the legend to Fig. 1

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40 30 20

+

10 0

Control

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The changed sensitivity to DCCD is of significance when trying to detect changes in the inhibitory effects of different antibiotics on E. coli. This might be due to the role a membrane plays in bactericidal effects of tetracycline and chloramphenicol [32–36]. These antibiotics were effective in decreasing E. coli K12(k) specific growth rate: the effects were obtained in a concentration-dependent manner (for 2–10 lM, not shown) as determined before by different groups [33, 37]. However, the EMR at the frequency of 51.8 GHz changed the following growth of bacteria in the presence of these antibiotics: the prolong lag-phase duration and the decreases in specific growth rate (P \ 0.025 for the growth with tetracycline) were determined (Fig. 3). It should be noted that the EMR of the mentioned frequency was used to define effects for antibiotics, since EMR at the frequency of 53 GHz was stronger to inhibit E. coli growth (Fig. 1) and to suppress DCCD-inhibited H+ efflux and ATPase activity (Fig. 3).

53 GHz

Fig. 3 The inhibitory effect of DCCD on H+ efflux (a), the change in H+ flux (b) and in DCCD-inhibited ATPase activity (c) for E. coli K12(k) after radiation with EMR of the frequency of 51.8 and 53 GHz. The radiation time was 60 min, grown cells were transferred

B

Control

51.8 GHz

-1

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

-1

+ 1 mM DCCD

Inhibitory Effects of Antibiotics on Bacteria and EMR

(nmol Pi.min .mg protein )

50

Furthermore, a significant decrease in DCCD-inhibited ATPase activity of E. coli membrane vesicles after exposure of bacteria with EMR at the frequency of 51.8 or 53 GHz was determined in K+-free (concomitant K+ was of *0.02 mM) and K+-containing (100 mM K+) assay medium, both (Fig. 3c) confirming a target role of F0F1 in bacterial action of EMR. It should be noted that DCCD inhibits the F0F1-ATPase activity since there is no DCCDinhibited ATPase activity in E. coli atp mutant with deleted F0F1 [22]. Moreover, the result was obtained in the K+-free and K+-containing assay medium when the difference between DCCD-inhibited ATPase values for these mediums indicates its dependence on K+. The latter could be explained by an association of the F0F1-ATPase with K+-uptake (TrkA) system in E. coli (for review, see [31]).

DCCD-inhibited ATPase activity

+ 0.5 mM DCCD

12

+ 0.1 mM DCCD

60

A

H efflux (mMol/min/10

DCCD-inhibited H+ flux (%)

70

cells)

shown before [10, 12, 14, 15]. The resonance, in which proteins with own dipole moments can be entered, might be a basis of membranous [10, 14] and other [1, 2, 14, 17, 18] mechanisms in the action of extremely high frequency EMR on bacteria. The oscillation with bacterial DNA and the other macromolecules or their parts is not ruled out (for review, see [16]). Interestingly, the H+-transporting F0F1-ATPase might play a key role in membranous mechanisms of EMR effects, which are followed by various sensitivities of exposed bacterial cell to reagents like DCCD, an inhibitor of F0F1. Indeed, the inhibitory effect of DCCD (in concentrations of 0.1–1 mM) on energy-dependent H+ fluxes by E. coli decreased after radiation of bacteria with EMR at the frequency of 51.8 or 53 GHz (Fig. 3a): DCCD-inhibited H+ fluxes (see: Materials and Methods) by the exposed cells were low in comparison with those in control cells (P [ 0.1 for the difference of the values for DCCD-inhibited fluxes by exposed cells at the frequency of 51.8 GHz in comparison with that of control cells for 0.1 mM DCCD; P \ 0.05 for such a difference for 0.5 mM DCCD) despite different values of H+ fluxes (Fig. 3b). Less sensitivity to DCCD could result from the conformational changes in the subunits of the F0F1-ATPase [27]. Note that DCCD is known as a non-specific inhibitor of the F0F1-ATPase, which is also able to block cytochrome bc1 complex and cytochrom c oxidase, components of the electron transfer chain in bacterial or other coupling membranes (for review, see [28]). However, DCCD has been clearly shown to inhibit H+ flux through the E. coli F0F1-ATPase under fermentation conditions [19, 29, 30]. This is in favor with the fact that H+ efflux by E. coli could become DCCD-resistant in some atp mutants with defects in the F0F1-ATPase or with deleted F0F1 [22, 29, 30]. Moreover, DCCD inhibits F0F1 in E. coli owing also to the absence of cytochrome bc1 complex and cytochrome c oxidase (for review, see [31]).

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 -0.05 Control -0.1 -0.15

C + K+ - K+

51.8 GHz

53 GHz

53 GHz

into the appropriate assay medium (see Materials and methods), and glucose (20 mM) was added; for ATPase assay, 3 mM ATP (Tris salt) was added into the assay medium with membrane vesicles (c). For the others, see Materials and methods and the legends to Fig. 1


The results with E. coli K12(k) (Fig. 3) are mentioned to confirm the input of membranous changes in the bacterial action of low intensity extremely high frequency EMR, when the F0F1-ATPase is playing probably a key role, although the action of this EMR is also offered on the structure and properties of water [13, 17] (for reviews, see [1, 2, 16]) leading, for instance, to increase in the chemical activity of water or hydration of proteins and changes in other cellular structures. These could also relate to modifications of structure, stability, interaction with each other, enzymatic activity, and function of membrane proteins, namely, of the F0F1-ATPase. The changed DCCD-inhibited H+-efflux through this ATPase and its ATPase activity confirm the action of low intensity extremely high frequency EMR on F0F1. So, the a target role of F0F1 is suggested for the effects of EMR and this might be following the changes in properties of water molecules and membrane proteins. Such a role of F0F1 is likely since this is the main protein present in a higher amount in the E. coli membrane [38–40] and associates with secondary solute transport systems and/ or enzymes of anaerobic oxidation-reduction (for review, see [31]), and therefore, has significance in the habitability of fermenting bacteria. In all cases, the changes in the activity of the F0F1-ATPase point out the membranous effects of low intensity extremely high frequency EMR on bacteria, which are similar to those in other cells and organisms as well as to the effects on lipid bilayers with their changing volume or permeability (for a review, see [1]). The effects of such EMR can be a result of resonant interaction with proteins and other cellular components and

1.1

1.6 1.5

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1.4 0.9

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Chnage in duration of lag-phase

Discussion and Concluding Remarks

structures, when in case with E. coli, it is shown that, regardless of the intensities of extremely high frequency EMR, resonance is at the frequencies of 41.5, 51.8, or 70.6 GHz [18]; other resonant frequencies are not ruled out. In order to explain the mechanisms for the bacterial effects of extremely high frequency EMR with low-intensity, quantum-mechanical approach and physics of nonequilibrium and nonlinear systems are considered and a model is suggested for the interaction of EMR with genome (for reviews, see [1, 2, 16]); however, the effects of EMR on E. coli and other bacteria, especially membranous effects and primary cellular mechanisms of these effects, require a further study. All these changes in membrane transport proteins and enzymes and in genome mentioned can lead to inhibition of bacterial growth and survival. Besides, it is of interest that these effects of EMR were observed with bacteria grown till the stationary growth phase although weak response to EMR was reported before with exponentially grown cells [11]. This can be interpreted by differences in bacterial membrane structural organization in stationary phase grown cells when differential effects of external probe absorption on the membrane and of transport across the membrane have been determined [19, 25]. Furthermore, tetracycline and chloramphenicol affect E. coli growth and survival and these effects could be enforced by EMR of extremely high frequency (Figs. 2 and 4). It is assumed that the latter might change antibiotic sensitivity or resistance. It is of interest that action of EMR

Change in specific growth rate

Furthermore, this frequency was mentioned [18] to be resonant for E. coli and might have a distinguishing effect. EMR enforced effects of antibiotics on bacterial survival too: such an effect’s validity was shown for the 1st day (Fig. 1). This could be due to lower survival observed for the 3rd and 4th days (Fig. 1) or to some compensatory or repair mechanisms suggested for bacteria [12, 14] and to miscellaneous defensive proteins like those discovered in animal cells [12]. Note that the concentration of antibiotics used (4 lM, Figs. 1 and 3) was close to the minimum inhibitory concentration shown by different authors [33, 37]. Thus, in addition to sensitivity to DCCD, EMI at the frequency of 51.8 GHz enforces the inhibitory effects of antibiotics on bacterial growth and survival. This seems to be in favor of the results showing that extremely high frequency EMR with low intensity could change the Staphylococcus sensitivity to various antibiotics affecting membranous properties [8].

101

0.6

Control

51.8 GHz

Fig. 4 The effects of EMR with the frequency of 51.8 GHz on the E. coli K12(k) growth lag-phase duration and specific growth rate in the presence of different antibiotics. 4 lM tetracycline (Tet) or 4 lM chloramphenicol (Cmp) was added to the growth medium. The lagphase duration and specific growth rate without radiation (duration of 60 min) was 1; for the others, see the legends to Figs. 1 and 2

102

[10, 14] and these antibiotics [32–36] on bacteria occurs through membranous mechanisms: changes in the properties of membrane proteins and namely in the F0F1-ATPase can mediate such action. Differences in membrane proteome [41], membrane permeabilization, discharging protonmotive force [32, 36], which is generated by the F0F1ATPase under fermentation conditions (for review, see [31]), disturbances in H+, K+ and Na+ transport [35], alterations in appropriate transport systems, and lowering ATP level [32, 34] increase sensitivity to antibiotics. In addition, alteration in DNA gyrase and other changes in gene expression or protein synthesis are also important in sensitivity to antibiotics (for review, see [42]). However, mechanisms for antibiotic sensitivity as well as of combined action of EMR and antibiotics are complex and not clear yet. The effects of extremely high frequency EMR are of significance to understand the distinguishing role of bacteria in biosphere leading to changed metabolic pathways and, for instance, to antibiotics resistance. Acknowledgements This study was done within the framework supported by Ministry of Education and Science of the Republic of Armenia (Grants # 0167-2005 and # 1012-2008).

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