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Pocnnttnrp Fffoct of Microwaves on the Genome Conformational State
of E. coli Cells
Igor Ya. Belyaev. Yevgeny D. Alipov, Victor S. Shcheglov, and Vltaly
^' J:
ys
J?
OV
Moscow Engineering Physics Ins^
u'^a^
ir
,skoyC^°f
fiV
’
"
(Temporary Research Collective “Otklik
, Vladimirskaya st.. 61
B
,
Kiev. GSP. 252017. C.I.S.
' Z. Naturforsch. 47c, 621 -627 (1992); received September 21, 1990/January
l, 199
Cellular Biology, Microwave Bioaction, Radiation Damage, Repair
Introduction
At present a
significant body of evidence has
been collected on the ability of microwaves in the
millimeter range to bring about biological effects
including those on the cellular level
[1.
2].
It has
been
found
that microwaves can
influence
the
processes of gene expression [3-5]. The
specific
features of such interaction are dependence on fre-
quency
and
also
effectiveness
of low
intensity
microwave radiation which does not result in sig-
nificant heating of the irradiated object. One of the
possible explanations of these
facts accounts for
the influence of millimeter waves on the genome
conformational state
[6]. The genome conforma-
tional state (GCS)
is expressed as the space-topo-
logical
organization
of
the
entire chromosomal
DNA, which
is ensured, among other things, by
the supercoiling of DNA and DNA protein bonds.
The GCS changes play a significant role in all ele-
mentary genetic processes - transcription, replica-
tion. repair.
The hypothesis which accounts lor the influence
of millimeter radiation most evident in the case of
stressed systems [1.7] among them bioobjects sub-
Reprini requests to
I. Ya. Belyaev. Moscow Engineering
Physics Institute. Kashirskoye sh..
-'1. Moscow. 115409.
C.l'.S.
Verlag der Zeitschrift fiir Naturforschung.
O_W-7400 Tubingen
0939-5075 92/0700-0621
$01.30/0
jected to ionizing radiation [6] has repeatedly been
verified.
The influence ot millimeter waves on the process
of the GCS repair after E. coli K 12 cell exposure
to X-rays was examined in this work. As a test for
appearance and repair of changes in a chromoso-
mal DNA we used the method of the anomalous
viscosity time dependencies (AVTD) in cell lysates
[6],
Materials and Methods
Microwave and X-ray irradiation
A block diagram of the experimental unit used
for microwave
irradiation
of
cell
suspension
is
given in Fig.
1. A G 4-141 generator served as the
source of extremely high frequency electromagnet-
ic radiation (EHF EMR). In the course of irradia-
if ififh
JL
-
-L
u
Fie
i
Block diaeram for microwave irradiation of cell
suspension;
1 - EHF EMR generator; 2 - controlled at-
tenuator: 3. 6 - directional coupler; 4 - frequency ana-
lyzer- 5 - measurement line (VSWR-meter); 7 - power
meter: 8 - pyramidal horn: 9 - cell suspension.
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, V
Belyaev „ a/.
Resonance Effect of Microwaves on .he Genome Confonna.iona, S.at. of E. col, CeUs
tionfhe^noya^^we--
^e
S,^^S.Uas.MH,=,of
-f
.0% -‘=*^2:.^ Si„?:™“ SIcoSy, <2%, and LET-
: rr^^a“!
5S”dy":
Petri
dishes. 50 mm in diameter, by means of a
tween addttion
of eaen
g
^ ^ ^£T papa j n
pyr^:"^“^erdS,,y
S^Tm"diP* EHF^
30 C for 40 h. af,.r .ha, ,he AVTD were mens-
irradiation frequencies used the local PD values at
ured.
the surface of the suspension differed by nearly an
order
of magnitude.
But
frequency changes
o
Method ofanomalous viscosity time dependencies
± 200 MHz did no. lead » signif-n. change »
^ ^^ o„ (he fact lhat
in soiu
the pattern of PD distribution. At the same time
frequency changes in a wide range (of the order of
units GHz) could lead to a marked displacement
of PD minima and maxima up to their inversion.
In the event of parity of output power in the wave-
guide. the PD value, averaged over the whole sur-
face under irradiation, did not change.
The specific absorption rate (SAR) was ™eas ‘
ured in two ways: by the acoustic method [9] and
the calorimetric method. The suspension tempera-
ture was measured by a microthermocouple.
Cells were subjected to X-rays (XR) using a ra-
tions of high-polymer DNA. placed
in
a rotan
viscosimeter, radial migration of DNA. which is a
directed movement
of macromolecules towards
the inner cylinder of the viscosimeter
(rotor),
is
observed [llj.
Measurements were carried out m a rotary cy-
lindrical viscosimeter with an automatic record of
the rotor’s rotation period [6]. In the unit used, the
rotor was set in motion by a constant moment ot
force created by an external electromagnetic field.
Upon completion of the lysis the rotor was sus-
*
.
r*
i.mnta avaminm
Cells were subjected to X-rays (XR) using a ra-
^ ^ meniscus of the
iysate examined,
diological unit RUP-150. The distance from
Therea fter the lysate was placed in a thermostati-
focus ,o .he s“s^
ns
|i
°n
v
wa
d
S
0" C”/oT Gy;min.
cally cou,rolled (30 ^C) jacke. of the viscosimeter
Microwave
3^
and X-irradiation of cells was carried
for^h
e“U
electromagnetic
field
is
out at ambient temperature.
switched on. the rotor starts moving. In the initial
stage of measuring the rotor’s rotation period (T),
Preparation of bacterial cells for experiments and
CS
TheT following
strains were used
in the^
E. coli K 12: AB
1 157
F
thr-I
ara
curve ^
After the DNA macromolecules had de-
proA2 lacGI tsx-33 supE44 galK
hisG
ited on the external surface of the rotor the ve-
mgl-5 1
rpsL 3 1
xyl-5
mtl-I
argE 3
hi- 1
\_ rac
P
Qf^ rotation decreased to the value typical
G62 F
+ proA23 lac-28 trp-30 his-51 rpsLR and
y
solvent , The dependence of the rotor's
also strain RM 1 17 which
is isogenic with strain
Qn
d in the cell lvsate on the time after
AB
1 1 57. Cells were cultivated by standard meth-
rotatlon starts
(t)
is called anomalous
ods in Luria broth or minimal medium M-9 [10],
tne
dependence.
The E. coli cultures used m the experiments were
sh
*
uld be noted here that AVTD cannot be
kept in spreadings on the Hottmger nutnen
ag
observed in protein solutions, because radial mi-
at 3-4 C C.
,
.
,
.
o
„r ,. tirm doesn’t take place
in solutions of mole-
Before irradiation. cell*. from the
.ugh. ool.ur
^
^
^ (han
D [1 !]. The param-
ai3U ouum
’
,
,
AB 1157. Cells were cultivated by standard meth-
ods in Luria broth or minimal medium M-9 [10].
The E. coli cultures used in the experiments were
kept in spreadings on the Hottinger nutrient agar
Before irradiation, cells from the night culture
were resuspended in concentrations of 3 — 9 *
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623
„
Pffpct of Microwaves on the Genome u
I. Y. Belyaev et al.
• Resonan
f„„, 1Pnnv band of 51.60—
t
of the AVTD curve in the cell lysate are de-
•
nM a
lQ^nciation with var
on the DNA nativity, DNA associau
c _rnteins
the microenvironment, etc. The ro
ious proteins,
\ which in this
tor's maximum rotation period (?««)
method is the most sensitive parameter character
izing the genome conformational state of
.
cells
was obtained from the AVTD curve- T
measurement error of the rotor's rotation period
was 2%
.
Results
c c
II rells with doses ot
iu
Irradiation of E- C°[\™"
TD curve of the cell
Gy leads to change
cauSe Qf these
lysate (Fig.
2, curve 2). The majo
changes is the considerable grease of T,
Aft.
pos,-irradiated
cell meuba on fcrJO^^
depending on the dose
f
curve (Fjg
2 .
complete
recovery
ns that during this
CUrVCA'°G <bs of"thl irradiated cells returned to
period the GCS
sense that we use the
the control leve
.
It is in
t
ional state .
cells were exposed
with-
frequency sw, 'c ”'';' 40 MHz during 30-90 min.
in the range of about -UU
cell
Fig. 2 (curve 4) shows the AVTD curve
VJWMW..—
—
irradiation within the
51 ’“'
51.78 GHz
microwaves in this range ef-
It can be seen that micro
To assess lhe
Sirs"r
r£V
ar“ r
irradiation. we used the follow,ng ratto.
_ f
v-'~ ^nian dl
^
UXRM-^XR
* f
- the average maximum rotor's rotation
peSd ta the lysates of cells lysated immedtately
after X-irradiati™
riod
in
the
lysates of cells lysated
after
X-irradiation and subsequent incu
scion
-
f
- the average maximum r
PHF
period
fn
the
lysates of cells subject^ to EHF
EMR during the rad,aU°”:'”
h ““dependencies .on
effective microwave ranges th
P
frequency were determine^
^dialed with
In these experiments, cens
, tn 1S m
: n
microwaves of a certain frequ^
y
°"
value of
after X-irradiation. To
^
in
the
rotor s m
A.VTD measurements
each of the -*«££
determined by
the rang", neri.
51.62-51.84 GHz
(s.ram
Fiu
G 62
3 -
lion
2. Anomalous viMmUy ume^pendencies^o^o
g
Sf fflfc
® - '““ba-
under the influence of EHF LMK.
Fig.
. Frequency- de^^HF M^; n7
radiation-induced repair ol lhe °Cb
cells (20 Gy: 15 min. 3 mW-cm
).
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Meet of Microwaves on .he Genome Conformations. S.aie of JT.
Celis
624
I- Y. Belyaev
al.
• Resonance Effect of Mtcrow
rotation oeriod derived from AVTD
Table
I. Values of the maximum^rotor s
d after X-irradmUon
curves obtained in lysates of E-‘0£ ^tiOn with EHF EMR (200 qW/cm2
) in
(20 Gv), subsequent incubation or irradiation w
the course of incubation.
Control
7.0 ± 0.3
26.2 ± 1.0
14.0
123
12.8 ± 0.6
p < 0.0004
41.25
12.1
41.30
7.2
6.4
7.2
6.9 ± 0.3
p < 0.0001
XR
0.9
10.1
9.7 ± 0.4
p < 0.0002
+
41.35
10.1
EMR
9.7
11.2
11.0 ± 0.7
p < 0.0004
+
41.40
12.2
I
16.2
17.3
16.7 ± 0.3
p < 0.001
41.45
16.6
15.2
16.2
15 .6 ± 0.3
p < 0.0006
41.50
15.6
* Standard error.
1 17) and 41.25-41.50 GHz (strain AB 1157).
to °ri“of
00 MHz and resonance frequencies
£ 5U6 OHZ and 4U2 GHz resp^tjvely^to tH=
Sated by acoustic and calorimetric methods,
estimated by
22 mW/
respectively.
Sng of the Sll suspension, when irradiated,
did not exceed 1 "C. The;*^"^“^nd.
ied
W
at
l
pD=
<
^W^W/cm
2 with heating not exceed-
So^^.^rosnppressionofmnair
10 min).
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v. Belyaev -
• Resonance Effect of
-* Genome
625
processes. We also studied the deaden™ofstg
pression of radiation-induced GCS repair on FU
of the microwave exposure at the
.
auency. The power dependence of x
is shown in
Fig. 5. Starting with a PD of
1 ^QCS repaid ^
for 5 min significantly suppressed GCS repair.
Anointed out above. Fig. 3 shows a frequency
dependence within the **-**<*%£»*
RM
1 17 strain. But this microwave irradiation wa
effective in repair suppression for the other strains
used- AB1157 and G62. Altogether
P
ZL were carried out, each revealing statically
3S
suppression
of
repair
processes
by
microwaves at frequencies of this
An EHF EMR effect on the genome conform
tional state was also discovered in the case of in-
verse sequence of,“VT^hEH “eMr' at to
X-ravs. Irradiation of cells with EHF hMK aiinc
51.78 GHz frequency (that is close ^ato
r
nance) before X-irradiation prevented the process
of radiation-induced repair (Table II).
Table
II. Values of the maxl^^ ™t
°ffeCt^HF^M
R
od
i»
US.»iTSl/xR poor) on
E. coli RM
1 7 cells
.
Type of
fnm± SE
[SI
Significance level
as compared with
effect
XR +
I
Control
XR
XR +
I
EMR + XR +
I
17.1 ±0.9
6.9 ±0.1
p<0.04
p< 0.02
12.5 ± 1-4
7.2 ±0.2
p< 0.003
Discussion
it u
generally accepted
that biological mem-
It
is
generally
v hmical and electromag-
branes arere^rsofchemi
those
=5S^--s=?S
So^,"rs"
ttes d«Se frequencies of resonant interacuom
XT™number of model studies microwave
effects were detected that had been causmi by a
change in the ion membrane transport [13
15J.
cnange
iu
u„ATT1hrane" effects examined
But the microwave
membrane
.
’
did not depend on the EMR frequency and there
toe do n^pertnit explanation of the resonance ef-
feet on the processes of cell development and gene
expression.*!! appeared to us that a pronuig ex-
nlanation of these observations could be supphed
bv the notion of the role of the genome conforma
tional state in forming cell's res°na”“ '^Twe
the millimeter wave exposure. I" »<h“
assumed that parameters of the GCS,
.
- P
pological organixadon of^°™“”l™
A
;vent
tetmine resonance frequencies.
Ir such
the GCS would be sensitive to the effect
waves of certain frequencies.
foTy when
DNA
break is induced per E co/i
ready made it possible to assume that the AVI L>
is sensitive not only to damages of the sug
“XsphStonds of the DNA smmndary struc-
ture The AVTD sensitivity to other changes o
Tome information,
particularly those caurnd
hv DNA-protein bonds, was confirmed by the e
oerTents we carried out [1 6]. The results obtained
P
h
courSe of our work indicate that repair o
‘he genome conformational state of bacterial cells
after ionizing irradiation is highly sensitive to the
resonance effect of millimeter waves
The microwave effect discovered cannot be ex
pla^d by trivia, heating. This was borne out by
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on the Genome Conformational State of E. coh Cells
626
i Y
Belyaev
al.
• Resonance Effect of Microwaves
^ chromosome DNA [18.
many of the
results
2£/s A??
by 5 C for 10 min during th
P
FinaUy ,
changes of DNA
transcription. reph-
bation has no influence on t
face did
elementary gene
P
nation. Consequently, it
r,
,imi,s of »
»
the observed resonancesi (± 20
estabilization of
the
cell ^vef mo
i
i
[2-5], changes m
r»SN-y-
iS and in cell division
ll
’u
*
062) were^sensi-
prisingly, this effe^™a^ith resonance frequency
strains use
( ^ 51.62-51.84 GHz frequency
are subjected to EM
h means that a cell,
«ve to
ofthese strains are isogenic by
before X-irradiation. This
X-irradiated.
band. The fir
third strain,
it differs
irrespective of whether or not it was
& cej>
known markers. As to
number of markers.
retains the microwave
t0 stress that
from the P'=™us ““
h
*
ve „„ mutations in the
tain period. It is especially
gen„„e
For mshmc*. °62 « Xcetylase or other genes
this memory is realized at the leveUt.«
gme of acetylormtlune am ^ biosymhesis of ar-
conformational state. This
EMR effect on
whose pro uc
fore
P
are not auxotrophic on this
by the fact that after a 5
of qcS repair
ginme and
possible
that
structural genes
^ £muons zz&gzs: sz
^Tte'^iscovered
frequency
nJmof resonance
by
"rt fhHhich
-- **—
CMiStS- -dying the gen^pres-
wo* are in accord-
sion of repressed X-prophag
coincidence is
Th' 5““'
h
i
l models predicting the exist-
E coti cells [3, 5], In«•»> “position of
anee wi* «heP^
1 ”0
discre
PK resonance states
«
S3i£->Vj£ —"thef—W °f “
E. coll ceils p,
-fj- — —
f the supposition oi
;
-
t
s of discrete resonate
ssrjz^,^ssirss =£-,u
*•—-“ '
»=" T1 -----
‘
In general, a chain o
e
micro-
ome s role in gi
rules on helicity for tran
r=
f Slmwi,,hem»depuh„ca.a tater
dat"
where DNA is attach
f dNA Qr its se-
Acknowledgements
V:
r * *
isrs
0nS„“"Suh'”' possibility .that the pri-
and S. PJWto for dismissing the re
mary^argets of
SSSST^-
teins, which take part in maintaining
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-
I. Y. Belyaev et al.
• Resonance Effect of Microwaves on the Genome Conformational State orr.-^ 'Cells
[1] W. Grundler, U. Jentzsch, F. Keilmann, and V. Put-
terlic, in: Biological Coherence and Response to Ex-
ternal Stimuli (H. Frohlich, ed.). PP- 65-85, Sprin-
ger Verlag, Heidelberg 1988.
[2] E. Postow and M. L. Swicord, in: CRC Handbook
of
Biological
Effect
of
Electromagnetic
Fielas
(C. Polk and E. Postow, eds.), pp. 425-460, CRC
Press, Inc., Boca Raton 1986.
[3] S. J. Webb, Phys. Lett. 73 A, 145 (1979).
[4] F. Kremer, C. Koschnitzke, L. Santo, P. Quick, and
A. Poglitsch,
in: Coherent Excitation in Biological
Systems
(H.
Frohlich and
F. Kremer,
eds.),
pp.
10-20, Springer Verlag, Berlin 1983.
[5] K.
V. Lukashevsky and
I. Ya. Belyaev, Med.
Set.
Res. 18,955(1990).
[6] Ye. D. Alipov,
I. Ya. Belyaev, D.
I. Yedneral, D. M.
Izmailov,
K.
V.
Lukashevsky,
L.
K. Obukhova,
O. V. Okladnova, and V. S. Shcheglov, in: Proceed-
ings of the Workshop on Genetic Effects of Charged
Particles (K. G. Amirtaev and S. Kozubek, eds.),
pp. 150- 160, JINR, Dubna 1989 (in Russian).
[7] S. P. Sitko, E. A. Andreev, and I. S. Dobronravova,
J. Biol. Phys. 16, 71 (1988).
[8] G. T. Butkus, K. K. Mikalauskas, and A. S. Pausha,
in: Medicobiological Aspects of Millimeter Radia-
tion (N. D. Devyatkov, ed.), pp. 230-234, IRE Ac.
Sci. U.S.S.R., Moscow 1987 (in Russian).
[9]
I. G. Polnikov and A. V. Putvinsky, Biofisika 33, 893
(1988) (in Russian).
(10]
J. Miller, Experiments in Molecular Genetics, p. 373,
Mir, Moscow 1976 (in Russian).
[1
1]
E. L. Uhlenhopp, Biorheology 12, 137 (1975).
1121 V. D.
Iskin, Yu. V. Zavgorodny, N. M. Yatsenko,
L. K.
Silina, E. V. Stepula, A. W. Medvedovsky,
B. G. Rice, and S. V. Rudenko, Biological Effects of
Millimeter Waves.
Review.
A.
Deposited Manu-
script,
p. 75, All-Union
Institute of Scientific and
Technical
Information
of
the
U.S.S.R., Moscow
1987 (in Russian).
[13] V. M. Shtemler, in: The All-Union Symposium with
Foreign Participants: Theoretical and Applied As-
pects of Usage of the Millimeter Electromagnetic Ir-
radiation in Medicine. Abstracts (S.
P. Sitko, ed.),
pp.
1 14-115, TRC “Otklik”, Kiev 1989 (in Russian).
[14] R. N. Khramov, Ye. M. Kobrinsky, A- K. Filippov,
and N. I. Porotikov, ibid., pp. 49- 50 (in Russian).
[15] I. Yu. Petrov and O. V. Betsky, Dokl. Acad. Nauk
U.S.S.R. 305, 474 (1989) (in Russian).
[16] Ye. D. Alipov, I. Ya. Belyaev, V. P. Kryuchkov, and
V. N. Lystsov, in: Proceedings of the Eight Balkan
Biochemical and Biophysical Days, pp. 1-2, Insti-
tute of Atomic Physics, Bucharest 1990.
[17] I. Ya. Belyaev, Ye. D. Alipov, V. S. Shcheglov, K. V.
Lukashevsky, and V. N. Lystsov, in: Proceedings of
Tenth
International
Biophysics
Congress,
p.
549,
Vancouver 1990.
[18] S. P. Sitko, V.
I. Sugakov, Dokl. Acad. Nauk Ukr.
S.S.R. 6B, 63 (1984) (in Russian).
[19] F. Keilmann, Z. Naturforsch. 41c, 795 (1986).
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I
f.
* 4
4*
t
PH
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376
R. E. Tigranyan and V. V. Shorokhov
7. Molecular-Bioiogical and
Cytological Aspects of Lympholeukaemia
(in Russian) (Eds
v
Nikolayeva and others) Nauka, Moscow, 1981
Russian)
(Eds. N
,
' SnoyaKM985
V ' * ^ MathematicaI Biophysics <in Russian > PP- 50-59, Kras. State l>„
|
Biophysics Vol. 33, No. 2, pp. 376-377, 1988
Primed in Poland
0006-3309/88 510.00
.
.
© 1989 Fersamon Pm>
-
FREQUENCY RANGE OF THE AUDITORY
EFFECT OF U.H.F.*
R- E. Tigranyan and V. V. Shorokhov
Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino (Moscow Regie.)
( Received 5 August 1986)
aCOUStlC Sl8nal fr°m an e,ectrod -vna'™emitter over the
SSTSit ^the zeroL^i!^
the firSt time in a
experiment.
I.
is
sequence of J h f
5°
?
C° UStlC ‘°nal S,
'
gnaI and ,he first harmonic of the pulse
sequence of u.h.f. are fixed most clearly at the points corresponding to low threshold value
in the threshold curve of the u.h.f. auditory effect.
terminati°n °f the boundaries of ^e frequency range of radio
.and lw'T!
y
u
hUmanS 11 WaS Sh°wn that ft is ,imited downwards by the value 8 kH:
.-
“pwa
J
ds by the mtrinsic high-frequency boundary
of hearing
(h.f.b.h.)
of
the
subjects. However, our findings
[3, 4] obtained in various physical models pointed k
possibility,
in principle, of human perception of the whole sound range and no
m
P
,
ar
.
°
U '
n Partlcular> this followed from experiments on a two-contour resonant
model based on the most fundamental principles known from the theory of hearinf
I
tW°'C°nt0Ur m0deI
fu]ly
reflects
the
mechanisms
of
hearing
aiw
he existing analogues of these mechanisms, isolates the first harmonic *
f*
tk
I*™!
Wlth°Ut Conflicting with the Physiological and neurophysiologic*
e abseBCe of residual sound [2] in the preresonance region in the nature
experiment
[1 ] may be explained by the following factors.
I’ ^fficient attention of the subjects during the experiment to establish
the
pre-
sence of beats at low frequencies.
2. The high level of noise (~40 dB above the hearing threshold) in a room where the
natural experiment was run.
3 The low amplitude of the frequency of the beats at the mean level at low rcpc'’
tion frequencies of the u.h.f. pulses.
4. The insufficient power of the u.h.f. pulse taking into account the fact that with &
in the repetition frequency of the u.h.f. pulses the threshold rises.
»
* Biofizika 33: No, 2, 349-350, 1988.
> nse m power in
t
tthe beats in the p
i level (~20 dB
Jar waveguide of
i density of the
flu:
[*of the head was irh
Jses chosen was
ui
earphones of a G
he frequency of the to
. being controlled w
ta repetition frequenc
(Subject. Starting fro:
tv
.
,
increase in the repe
erceived over the w
Sc tonal signal in o;
,
r perception in the opi:
frequences were 3-58,
-
®nd for the third 3-80,
i
iffy
—
f
fccorded zero beats in a ton.
(AZHELOV, V. V., et al.
'UTEN, J. F„ Proc. K
KAN, R. E. and St.
j? In: Biological Effect o
is..
Russian) ONTI NTs!
33 . No- 2, PP- 377-382, IS
DISTRIBUTION
m
Institute of Molecula
stion of the reasons foi
^ considered. Analysis of
indicate that the difference
fizika 33; No. 2, 351-