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Combined Microwave and Electron Beam
Exposure Facilities for Medical Studies and
Applications
Diana Martin1, Sabin Cinca2, Irina Margaritescu3, Monica Neagu4,
Nicusor Iacob1, Daniel Ighigeanu1, Constantin Matei1, Gabriela Craciun1,
Elena Manaila1, Doru Aurel Chirita3 and Mihaela Moisescu5
National Institute for Lasers, Plasma and Radiation Physics, Bucharest, Romania
*
[email protected]
2
Oncology Institute “A. Trestioreanu”, Bucharest, Romania
3
Military Clinical Hospital “Carol Davila”,Bucharest, Romania
4
National Institute “Victor Babes”, Bucharest, Romania
5
University of Human Medicine and Pharmacy ”Carol Davila”, Bucharest,Romania
1
The paper presents two radiation exposure facilities (REFs) which permit separate and simultaneous irradiation with microwaves (MW) of 2.45 GHz and electron beams (EB) of 6.23 MeV for
malignant melanoma (MM) cell investigations, in vitro (MW+EB-REF-vitro) and in vivo (MW+EBREF-vivo). The REFs are specifically designed for the following medical studies: 1) The effects
of separate and combined (successive and simultaneous) MW and EB irradiation on the B16F10
mouse - MM cell cultures without/with drugs incubation; 2) The effects of separate and combined MW and EB irradiation on human blood components irradiated in samples of integral blood from
healthy donors and from donors with MM; 3) The effects of separate and combined MW and EB
whole body irradiation on the C57 BL/6 mice bearing MM without/with drugs administration. Several representative results obtained by experiments with REFs in vitro and in vivo are discussed.
The most important conclusion of the experimental results is that low dose-total body MW+EB
irradiation combined with drugs administration could present a valuable potential for an advanced
study in malignant melanoma therapy.
Submission Date: 18 August 2008
Acceptance Date: 15 May 2009
Publication Date: 28 July 2009
INTRODUCTION
In addition to routine conventional radiotherapy
techniques [Olofsson, 2005], electron beams
(EB) are presently used or are under study for
cancer specialized therapies such as intensity
modulated radiation therapy and total body
Keywords: microwave, electron beam, combined
exposure, B16F10 cell, C57 BL/6 mouse
Guest Editor: Dr. Satoshi Horikoshi, Tokyo University of
Science, Chiba, Japan
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electron irradiation [Leung, 1998, Shouman, 2004,
Margaretic, 2002, Van Dyk, 1986]; irradiation
of blood and blood components [Moroff,
1997, Buston, 2000]; virus inactivation and
vaccine preparation [Smolko, 2005], and others.
Microwaves (MW) are presently used or are
under study for therapeutic applications in
areas such as cardiology, urology, surgery,
ophthalmology, cancer therapy, and for
diagnostic applications in areas such as cancer
detection, organ imaging, and more [Rosen,
Journal of Microwave Power & Electromagnetic Energy ONLINE
Vol. 43, No. 3, 2009
2002]. Low-dose total body irradiation
(LTBI) with ionizing radiation is known for
its antitumor immune modulatory effects
[Safwat, 2003]. The fractionated whole body
low dose ionizing radiation (LDR) induces
immunomodulation: LDR exposure enhanced
the function of macrophages and responses of
CD8+T cells in C57 BL/6 mice [Pandey, 2005].
The effect of low dose ionizing irradiation on
the function of endothelial cells lining tumor
vessels was studied. Low dose irradiation
of the endothelial cells within tumors is
a key determinant of the effectiveness of
radiotherapy and may offer a new strategy to
increase gene and/or drug delivery to the tumor
[Sonveaux, 2002]. One of the major side effects
of chemotherapy in cancer treatment is that it can
enhance tumor metastasis due to suppression of
natural killer (NK) cell activity. The millimeterwaves’ (MMWs) irradiation (42.2 GHz) can inhibit
tumor metastasis enhanced by cyclophosphamide
(CPA), an anticancer drug [Mahendra, 2006].
CPA caused a marked enhancement in tumor
metastases (fivefold), which was significantly
reduced when CPA-treated animals were
irradiated with MMWs. MMWs also
increased NK cell activity suppressed by
CPA, suggesting that a reduction in tumor
metastasis by MMWs is mediated through
activation of NK cells [Mahendra, 2006].
Whole body hyperthermia (WBH), a procedure
in which the body temperature is elevated by
MW exposure to 41°C, has been investigated
as a treatment for cancer, most commonly as an
adjunct to radiotherapy (thermoradiotherapy) or
chemotherapy (thermochemotherapy) [Green,
1991, Katschinski, 1999, Hildebrandt, 2002].
The analysis of the reported data demonstrates
that the EB and MW medical application
results depend strongly on radiation nature and
their physical parameters, biological matter as
well as on used exposure system configuration.
Also, the reported data suggest that low dose all
body irradiation with ionizing or nonionizing
irradiation may enhance the tumoricidal effects
International Microwave Power Institute
of radiation or chemotherapy, overcome
acquired drug resistance and can stimulate
certain components of the immune system that
may aid in destroying cancer cells.
Malignant melanoma (MM) is one of the
most aggressive human cancers, as a tumor just
a few mm-thick has the potential to kill the host
in more than 80% of the cases [Timir, 2006].
Besides the surgical elimination of the primary
tumor, there is no other effective cure for MM
[Timir, 2006, Yang, 2007]. MM is resistant to
ionizing radiations as well as to conventional
chemotherapies [Timir, 2006]. The combination
of ionizing radiation with other therapies is a
promising strategy in cancer therapy [Yang,
2007]. In view of this argument we decided to
investigate the tumoricidal effects of combined
EB, MW and chemotherapy on the MM cells.
The main goal of this work was to design
appropriate exposure systems that permit
the use of combined effects of EB and MW
without/with the addition of drugs in two
models: in vitro, on the MM cell cultures, and
in vivo on the C57 BL/6 mice bearing MM, in
order to find more MM efficacious therapies.
EXPERIMENTAL INSTALLATIONS AND
PROCEDURES
Two new radiation exposure facilities (REFs)
which permit separate and simultaneous
irradiation with MW of 2.45 GHz and
accelerated EB of 6.23 MeV are carried out:
• MW+EB-REF-vitro for MM cells exposure
in vitro; • MW+EB-REF-vivo for MM cells exposure
in vivo.
The REFs are specifically designed for
the following medical studies: 1) The effects
of separate and combined (successive and
simultaneous) MW and EB irradiation on the
B16F10 mouse - MM cell cultures without/with
drugs incubation; 2) The effects of separate and
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combined MW and EB irradiation on human
blood components irradiated in samples of
integral blood from healthy donors and from
donors with MM; 3) The effects of separate and
combined MW and EB whole body irradiation
on the C57 BL/6 mice bearing MM without/
with drugs administration.
The EB effect is related to the absorbed
dose (D) expressed in Gray or J kg-1 and
absorbed dose rate (D*) expressed in Gy s-1
or J kg-1 s-1. The MW effect is related to SAR
(Specific Absorption Rate), which is equivalent
to D* and SA (Specific Absorption) which is
equivalent to D. The MW absorbed energy
depends strongly on the environmental factors
(temperature, humidity), volume, nature, initial
temperature and the geometrical configuration
of the exposed sample as well as on the MW
applicator type in which the sample is exposed.
Different sample volumes absorb different MW
power levels from the same offered MW power
in the exposure applicator [Persch, 1995]. In
this case SAR and SA values depend strongly
on sample properties and exposure geometry.
Therefore, we expressed them by W/sample and
J/sample, respectively. We have also determined
prior to our experiments, the dependence of the
absorbed MW power amount versus type and
exposure geometry of the samples used in the
experiments.
Both experimental facilities, MW+EB-REF-vivo
and MW+EB-REF-vitro, consist mainly of the
following units (Figures 1 and 2):
• An accelerated EB source: ALIN-10
electron linear accelerator of 6.23 MeV and
adjustable absorbed dose rate from 0.002 Gy s-1
up to 70 Gy s-1 (built in the Electron Accelerator
Laboratory of the National Institute for Laser,
Plasma and Radiation Physics, Bucharest,
Romania);
• A mechanical and electrical modified
microwave domestic oven (MEM-MWO) in
which are injected both EB and MW.
The commercial domestic oven was first
used in scientific experiments and remains
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Figure 1. The schematic drawing of the
MW+EB-REF (vitro or vivo).
Figure 2. Photograph of the MW+EB-REF
(vitro or vivo).
the basic design for the most sophisticated
models. We have been attracted to the use
of the domestic microwave oven due to its
simplicity of construction and adaptability to
many different loads. However, certain steps
were taken in order to produce an apparatus
with appropriate characteristics to biochemical
and biophysical research.
The first step was to properly modify the
magnetron power supply to ensure variable
magnetron output power. In our installations
the conventional operation of 2.45 GHz oven
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Vol. 43, No. 3, 2009
Figure 3. The schematic drawing of MEMMWO used with EB+MW-REF-vitro.
Figure 4. The schematic drawing of MEMMWO used with EB+MW-REF-vivo.
magnetron supplied by an L.C. single-phasehalf-wave doubler (L.C. HWD) was modified
in order to permit the use of a manuallycontrolled or PC-controlled electronic regulator
for the MW power adjustment [Martin, 2001].
The magnetron main power units consisting
of a high voltage diode (HVD), a high voltage
capacitor (HVC) and a high voltage anode
transformer (HVAT) are similar to the units
used for the conventional magnetron supplying
system. The difference consists in the use of a
separate transformer for the filament supply
(HVFT) and of a triac controlled regulator
(TCR) added to the HVAT primary circuit. The
microwaves are generated as 10 ms pulses at
50 Hz repetition rate.
The second step was to punch a rectangular
hole of 0.17 m x 0.17 m on the oven multimode
cavity upper plate and cover it with a 100 μm
thick aluminum foil. The ALIN-10 scanned EB
is perpendicular, and was introduced through
this aluminum foil into the oven multimode
cavity (Figures 1 and 2).
The third step was to modify the geometry
and rotation velocity of the sample rotary
system (Figures 3 and 4). The rotation velocity
can be modified from one rotation/1 s to one
rotation/20 s depending on the desired dose at
certain MW-SAR and EB-dose rates.
The MW+EB-REF-vitro permits simultaneous
exposure of 14 marked cylinders of PP T309 type
(made of polypropylene with silicone washer
seal and external threads) with cell culture,
arranged into a rotating cylindrical configuration
(Figures 3 and 5). For the experiments with
MW+EB-REF-vivo, the C57 BL/6 mouse,
placed into a special designed cylindrical cage,
was used (Figures 4 and 6). The C57 BL/6
mouse cage is made from a marked cylinder of
250 ml, PMP 2574 type cut at 112 mm from
its sole. Two Teflon pistons with aeration
apertures assure the mouse immobilization
during radiation exposure. During the radiation
exposure time the mouse cage can perform two
rotation motion types: in the horizontal plane
and around its axis. During one horizontal
rotation the mouse cage accomplishes two
axial rotations. Horizontal motion transmission
to the mouse cage is performed by a Teflon
arm fitted to the upper end with an aperture
in which a Teflon axle is rotating. A mounted
mouse cage is located on one Teflon axle end,
and a Teflon friction wheel on the other axle
end. The friction wheel is in permanent contact
with a fixed platform that generates the cage
axial rotation. The desired radiation exposure
homogeneity of PP T309 cylinders or the C57
BL/6 mouse cage is obtained by presetting the
exposure time so that each sample performs
one, two, three or more complete rotations
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43-3-15
Figure 5. Photograph of the MW+EB-REF- vitro.
Figure 6. Photograph of the MW+EB-REF- vivo.
inside the MEM-MWO multimode cavity
during irradiation process. The sample motion
starts and interrupts simultaneously with the
MW and/or the EB switch on and switch off,
respectively.
8 and 3.6 at 24 h, 48 h and 72 h, respectively. This
is an effect of drugs uptake stimulation by MW
exposure. This demonstrates that, as in the case
of the combination of a cytotoxic drug with
cells membrane permealization by high voltage
electric pulses [Mir, 2000, Kotnik, 2000,
Gothelf, 2003], the MW exposure is able to
increase the delivery of drugs into living cells
only at high SAR values that is at high electric
field strength values of the MW electric field
component.
Figure 9 presents effects of the separate
EB, separate MW and simultaneous EB+MW
irradiation on the B16F10 cells survival fraction
(irradiated sample/control sample). The maximum
reduction of the B16F10 cells survival fraction,
by a factor of 8, is produced by simultaneous
MW+EB irradiation (2 Gy +12.5 W/sample).
The investigation of the effects of the
EB, MW and EB+MW irradiation modes on
the blood components was performed using
samples of 1.5 ml integral blood obtained by
vein puncture from 33 patients: 26 patients
with malignant melanoma (MM) of different
stages (I-IV) and 7 healthy volunteers. The
studies were focused on the concentration of
protein fractions and proteins with enzymatic
activity. In the used range of irradiation parameters
(2-6 Gy for EB and 5-25 W/sample during several
seconds for MW) the protein fractions suffered
little changes, though enzymatic proteins were
RESULTS AND DISCUSSION
In vitro results obtained with EB and MW
exposure
The investigation of the effects of the EB, MW
and EB+MW irradiation modes on the B16F10
mouse melanoma cells culture and on the
human blood components (proteins and cells)
was performed using the MW+EB-REF-vitro.
The reduction of tetrazolium salts (MTT test)
was used to examine B16F10 cell viability [Van
de Loosdrecht, 1994]. Several representative
results obtained by experiments in vitro
performed with the MW+EB-REF-vitro are
presented in Figures 7-12. The absorbance
(which is the cell’s viability, evaluated by the
MTT test) decreases by a factor of about 1.3
at 24 h, 48 h and 72 h after MW exposure of
B16F10 cell culture samples without DAC
(Figure 7). At a sufficiently high SAR level
(20.5 W/sample) (Figure 8) and appropriate
MW exposure times that overcome the
temperature rise over 38°C, MW increases the
DAC cytotoxicity versus SAR, by a factor of 2.8,
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Journal of Microwave Power & Electromagnetic Energy ONLINE
Vol. 43, No. 3, 2009
Figure 7. Absorbance (cells viability reduction)
versus SAR for samples without DAC.
Figure 8. Absorbance (cells viability
reduction) versus SAR for samples with DAC.
Figure 9. The effects of different irradiation
modes on the B16F10 mouse MM cell
cultures.
Figure 10. LDH versus different irradiation
modes for the R. O. volunteer with MM.
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43-3-17
Figure 11. LDH versus different irradiation
modes for the healthy S. M. volunteer.
Figure 12. The tritiated thymidine lymphocyte
proliferation tests.
notable affected. One of the most affected
proteins was LDH (Lactate dehydrogenase).
The enhancement of LDH enzymatic activity
depends on blood donor (healthy or with MM)
and on the irradiation modes. In the case of
MM volunteers, the experiments demonstrated
that the effects of different irradiation modes
on LDH depend also on the MM stages. In the
case of the blood MM donors (stages III and IV)
with initial high values for LDH (180-190 IU L-1),
radiation exposure produced a small increase of
LDH (up to 26%) while for MM donors (stages
I and II) with medium initial LDH values (140160 IU L-1), all irradiation modes induced
a significant increase of LDH (up to 44%).
Certain irradiation modes, especially MW
irradiation, produced blood hemolysis (BH)
for certain MM volunteers, but not for others.
In the case of healthy volunteers that had low
initial LDH values (45-100 IU L-1), separate EB
irradiation induced little change on LDH, while
separate MW irradiation and simultaneous
EB+MW irradiation greatly increased LDH
(up to 70%), without blood hemolysis. Only
high MW power/sample induces hemolysis for
healthy donors. Also, it is important to note that
the LDH growth by MW and EB+MW exposure
for healthy volunteers is more significant than
for the volunteers with different cancer stages.
Figures 10 and 11 give several demonstrative
examples for a MM volunteer (R.O.) and a
health volunteer (S.M.). The LDH increasing for
the healthy S.M. (Figure 11) is more significant
than for the R.O. with MM (Figure 10). In many
cases, EB addition to MW diminishes the MW
tendency to induce hemolysis and to increase
LDH (Figures 10 and 11). These experimental
results suggest that the LDH behavior in the
integral blood samples that were exposed to
different irradiation modes could be used as a
new diagnostic test that could help follow-up
cancer patients.
A very important test in cancer diagnosis
is the proliferating capacity of lymphocytes
in vitro, determined by the tritiated thymidine
lymphocytes proliferation tests (TTLPT). Radioactivity
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Vol. 43, No. 3, 2009
is measured with a β-counter and results as
pulses per min. (ppm). Ratio of ppm sample/
ppm control is named lymphocyte proliferation
index (LPI). Figure 12 presents the results
obtained for the MM patient R.O. (stage I).
A significant decrease in LPI was observed,
particularly for MW irradiated samples (by a
factor of 15 for MW of 6.5 W/sample). Although
separate EB irradiation also decreases LPI
with increasing dose, additional use of EB to
MW seems to diminish the MW tendency to
drastically decrease LPI.
In vivo results obtained with EB and MW
exposure
Forty-nine C57 BL/6 mice, weighing 20±2 g,
were divided into seven groups (G1-G7) with
7 mice in each group: G1 with healthy mice
and G2-G7 (randomly divided) with MMbearing mice. EB exposure consisted of 1 Gy
fractionated total body irradiation over 10
consecutive days (dose rate of 0.0022 Gy s-1)
without/with dacarbazine (DAC) administration
(80 μg/mouse/day) or without/with bleomycin (BL)
administration (4 μg/mouse/day). MW exposure
used in conjuction with EB was performed at
SAR=1.63 W/mouse and SA=74.98 J/mouse/day.
The EB separate exposure or simultaneous EB
and MW exposure (EB+MW) was performed
with MW+EB-REF-vivo during 46 s (two complete
horizontal and four complete axial rotations
of the cage with C57 BL/6 mouse). The
DAC or BL administration was performed
just before irradiation procedure. The mouse
groups received over 10 consecutive days the
following treatment type: G1(healthy group):
EB+MW; G2 (MM): EB+MW; G3 (MM):
EB+MW+DAC; G4 (MM): EB+MW+BL; G5
(MM): EB; G6 (MM): EB+DAC; G7 (MM):
EB+BL. Tumor growth was monitored by
measuring tumor diameters in two dimensions
with a caliper every other day. Tumor volume
was calculated as follows: [L (long diameter) × S2
(short diameter)]/2.
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The results are: in G1, G2, G4, G5, G6 and
G7 none of mice died; in G3 two mice died after
DAC administration; in G3 and G5 none of the
mice grew a tumor; in each G2, G4 and G5
one mouse grew a small tumor (<300 mm3); and
in each G6 and G7 two mice grew large tumors
(745-10301 mm3). It seems that the DAC or BL
addition to EB is not an appropriate procedure.
The preliminary conclusion is that total body
EB irradiation + total body MW exposure +
drug could be a novel MM therapy if EB, MW
and drug application sequences and doses are
optimized.
CONCLUSIONS
The main conclusions of our work are as
follows:
• A higher reduction of B16F10 cells in
culture could be obtained by MW+EB than
by separate EB or MW irradiation if EB and
MW application sequences and doses are
optimized.
• In certain cases, at sufficiently high SAR
levels and appropriate MW exposure times that
overcome the temperature rise over 38°C, MW
exposure increases the DAC cytotoxicity. This
is an effect of drugs uptake stimulation by MW
exposure.
• In many cases, in vitro experiments, the EB
addition to MW exposure diminishes the MW
tendency to induce blood hemolysis, to increase
LDH and to decrease lymphocyte proliferation
capacity;
• The human blood component’s behaviour
(especially LDH activity) in the integral blood
samples exposed to different irradiation modes
could be used as a new diagnostic test that, in
addition to other known tests, will help followup patients with MM;
• The EB+MW+Drug procedure is a
novel MM combined therapy that along with
other known combined MM therapies could
contribute if EB, MW and drug application
sequences and doses are optimized.
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