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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 43-3-12 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 43-3-13 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 43-3-14 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 Journal of Microwave Power & Electromagnetic Energy ONLINE 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 International Microwave Power Institute 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, 43-3-16 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. International Microwave Power Institute 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 43-3-18 Journal of Microwave Power & Electromagnetic Energy ONLINE 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. International Microwave Power Institute 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. 43-3-19 REFERENCES Buston, M. J., Cheung, T., Yu, P. K. N. and Stokes, M. J. (2000). “Blood irradiation with accelerator produced electron beams.” Phys. Med. Biol., 45, pp.139-142. Gothelf, A., Mir, L. and Gehl, J. 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