Influence of microwaves and electron beams on red blood cells
A. Hategan1, D. Martin1, C. Butan1, A.Popescu1 C.Oproiu1, V.V. Morariu2
1.Institute of Atomic Physics, Electron Accelerator Laboratory, P.O.Box MG36, Magurele, Bucharest, Romania
2. Institute of Isotopic and Molecular Technology, CP 700, 3400 Cluj-Napoca
ABSTRACT
The effects of 6 MeV electron beam ( 0o C) and of 2.45 GHz microwave (-196o C) at
0-850 W, as well as preliminary results of the combined treatment, on the osmotic fragility of
human erythrocyte membranes are presented. The changes in the properties of the erythrocyte
membranes were estimated by measuring the radiation induced haemoglobin release from the
erythrocytes (hemolysis) and the osmotic fragility of the membranes, determined by
postirradiation induced osmotic stress. We obtained no hemolysis induced by accelerated
electrons in the range 0-400 Gy, whereas the microwave irradiated erythrocytes showed in the
ranges (1-2 min) and (400-500 W ) values of very small hemolysis, down to 50% from the
control. The osmotic stress experiments indicated a significant increase in osmotic fragility
for 200 -400 Gy electron doses, whereas the 100 Gy irradiated sample showed a hemolysis
down to 35% from the control. Similarly, the microwave irradiated erythrocytes showed
values down to 60% from the control for (1min, 850W). Both radiations induced at definite
parameters values of very small hemolysis, suggesting a stabilisation of the membranes and an
increase in osmotic resistance. Our preliminary results on simultaneous irradiation of the
frozen erythrocytes seem to indicate a significant contribution of microwaves in hemolysis
evolution, while the successive irradiation procedure did not allow so far a clear
interpretation, further studies being necessary to elucidate the membrane molecular
mechanisms induced .
INTRODUCTION
The actual experimental data provided by the scientific literature concerning the
effects of microwave irradiation and combined microwaves with electron irradiation on the
biological samples are controversial and the molecular mechanisms involved in the physicochemical modifications that occur are not completely clarified [1]. Freezing of the samples at
liquid nitrogen temperature makes possible the determination of the non-thermal effects of
microwaves and the direct effects of electron irradiation on the biomembranes [1,5,6]. The
temperature variations in the frozen state of the sample do not affect the erythrocyte
membrane properties.
METHODS
Sample preparation
Human blood from a healthy donor stored for 3 days in a blood bank was
washed three times according to a standard procedure with phosphate buffer solution 150 mM
NaCl / 5 mM K2HPO4 / KH2PO4 at pH 7.4. Packed erythrocytes obtained by this procedure
were suspended at a final cytocrit of 16.66%. In the case of microwave irradiation, the packed
erythrocytes were suspended at a final cytocryt of 16.66% in a crioprotectant buffer solution
consisting of 28 g glicerol/2.8 g sorbitol/ phosphate buffer solution 150 mM NaCl / 5 mM
K2HPO4 / KH2PO4 at pH 7.4.
Irradiation procedure
Device: The samples were irradiated by using: a rectangular cavity (RC) specially
designed to permit either electron beam irradiation only, microwave irradiation only, or
combined (simultaneous or succesive) electron beam and microwave treatement. Microwave
power, up to 850 W at 2.45 GHz is coupled to the RC sidewalls via a slotted waveguide
system. Scanned vertical 5 MeV electron beam of a linear accelerator with the following
parameters: 5-10 MeV energy of the electron beam, average intensity of 5-10 uA, frequency of
the pulses 100 MHz, time of a pulse 5-10 us was introduced perpendicularly by the upper end
of 100 um thick.
Temperature: The samples were termostatated at 0o C using a ice-water bath, and at 196o C using a liquid nitrogen bath .
Dosimetry: The dose accumulated by the samples was estimated using Fricke
dosimetry in calibrating the monitor of the linear accelerator.The dose rate was estimated to
be constant during irradiation (2 kGy/min).
Hemolysis Measurements
 The irradiation induced hemolysis was determined by measuring the hemoglobin released
in the supernatant of the samples because of radiation action. This was achieved by
measuring the optical absorbency of the supernatant solution at 540 nm and the results
were reported to the total hemoglobin released by the control sample in the presence of
distilled water.
 The osmotic fragility of irradiated erythrocytes was determined by measuring
spectrophotometrically the hemoglobin loss in the supernatant of the erythrocytes exposed
to osmotic shock by suspending them in solutions with different concentrations of NaCl .
In the particular case of the microwave irradiated erythrocytes, the cells were washed to
eliminate the protector solution, by using a standard procedure which consists of several
centrifugations and resuspensions of the cells in buffer solutions of different osmolarities
Finally the cells were suspended in a buffer solution consisting of 150 mM NaCl / 5 mM
K2HPO4 / KH2PO4 pH 7.4, at a hematocrit of 6%, for each sample. The osmotic stress
was performed by suspending 300 ul of every 6 % solution in 5 ml of buffer solution
containing different NaCl concentrations (final cytocrit of 0.34 %). After 10 minutes
necessary for osmotic stress results to be complete, the samples were centrifuged at 500 g
for 5 min and the hemoglobin released in the supernatant determined
spectrophotometrically. All the results were reported to the value of the maximum
hemolysis obtained for the nonirradiated sample.
RESULTS AND CONCLUSIONS
Our results showed no hemolysis induced by electron irradiation at 0o C, in the dose
range 0-400 Gy, indicating that the membrane modifications due to radiation interaction do
not reach a critical point to cause swelling of the cells and consequent lysis.
The microwave irradiated erythrocytes showed in the ranges (1-2 min) and (400-500
W ) values of very small hemolysis, down to 50% from the control (fig.1).
The influence of radiation is tested in the osmotic stress experiments performed after
irradiation, which give information on the most resistant erythrocyte population in the
samples.
The electron irradiated samples showed a drastic behaviour in the osmotic stress
experiments (fig.2).They showed a hemolysis induced by the strongest osmotic stress with 7585 % higher than in the control sample. In the case of a certain electron dose (100 Gy), the
hemolysis degree was found much smaller than for the control sample (35 % from the control,
at the strongest osmotic stress).A similar behaviour of the erythrocytes was found in the case
of microwave irradiation of the erythrocytes at subzero temperatures.
The microwave irradiated erythrocytes at -196o C exhibited a similar behaviour with
the electron irradiated red cells (fig.3). The samples irradiated for (1 min, 850W) showed a
hemolysis degree of approximativelly 60 % from the control sample. In the case of microwave
irradiation it can be assumed that this effect is due to the non-thermal component of
electromagnetic radiation . Possible resonances at the molecular level also have to be taken
into account for the explanation of the results obtained: the points of very small hemolysis
might be seen as resonances at the molecular level, in response to irradiation time. They
should be characterised by a “stationary state” of the membranes, that implies an increased
resistance of the cells to osmotic stress.
The similarity between the results obtained in the case of electron irradiation at 0o C
and microwave irradiation at -196o C, might suggest similar energy absorbtion into the
samples,in the two different sets of irradiation conditions (liquid samples at 0o C and frozen
samples at -196o C). Much more experimental data is necessary in order to clarify this similar
behaviour, obtained for the two different radiations.
Our preliminary results on simultaneous irradiation of the frozen erythrocytes (fig.4
a,b) seem to indicate a significant contribution of microwaves in hemolysis evolution, while
the successive irradiation procedure (fig.5) did not allow so far a clear interpretation, further
studies being necessary to elucidate the membrane molecular mechanisms induced.
.
Figure 1. Hemoglobin loss from microwave irradiated erythrocytes at different exposure
times and different power levels.
0 mM NaCl
50 mM NaCl
60 mM NaCl
70 mM NaCl
80 mM NaCl
90 mM NaCl
100 mM NaCl
110 mM NaCl
120 mM NaCl
150 mM NaCl
electrons irradiation
200
180
160
Hemolysis (%)
140
120
100
80
60
40
20
0
0
100
200
300
400
500
600
Dose (Gy)
Figure 2. Influence of different electron doses on the
erythrochyte membranes.
150 mM NaCl
120 mM NaCl
110 mM NaCl
100 mM NaCl
90 mM NaCl
80 mM NaCl
70 mM NaCl
60 mM NaCl
50 mM NaCl
0 mM NaCl
120
100
Hemolysis (%)
osmotic fragility of human
80
60
40
20
0
0
1
2
3
4
5
6
Irradiation time (min)
7
8
9
Figure 3. Influence of microwave irradiation time on the osmotic fragility of frozen
human erythrocyte membranes.
22
20
MW+e-
18
MW + e-
e-
16
14
%
[
H
12
10
8
e-
6
0
20 40 60 80 100 120 140 160 180
Dose[Gy]
(a)
2
2
2
0
-
MW + e
1
8
M
W
+
e
M
W
H[%]
1
6
MW
1
4
1
2
1
0
8
6
4
0 2
0 4
0 6
0 8
0 1
0
01
2
01
4
01
6
0
Irra
d
ia
tio
n
tim
e
[s
]
(b)
Figures 4 a,b. Hemolysis induced by simultaneous microwave and high energy electron
irradiation of frozen erythrocyte membranes.
H[%]
2
0
1
8
M
W
1
6
M
W
+
e
,s
im
u
lta
n
e
o
u
s
1
4
e
,M
W
,s
u
c
c
e
s
iv
e
successive
1
2
successive
M
W
,e
,s
u
c
c
e
s
iv
e
1
0
8
6
4
02
04
06
08
01
0
0
1
2
0
1
4
0
1
6
0
1
8
0
2
0
0
Ir
r
a
d
ia
tio
n
tim
e
[s
]
Figure 5. Hemolysis induced by simultaneous and successive microwave and high energy
electron irradiation of frozen human erythrocyte membranes.
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