Delta radiomics analysis of Magnetic Resonance guided radiotherapy imaging data can enable treatment response prediction in pancreatic cancer.

BACKGROUND: Magnetic Resonance Image guided Stereotactic body radiotherapy (MRgRT) is an emerging technology that is increasingly used in treatment of visceral cancers, such as pancreatic adenocarcinoma (PDAC). Given the variable response rates and short progression times of PDAC, there is an unmet clinical need for a method to assess early RT response that may allow better prescription personalization. We hypothesize that quantitative image feature analysis (radiomics) of the longitudinal MR scans acquired before and during MRgRT may be used to extract information related to early treatment response. METHODS: Histogram and texture radiomic features (n = 73) were extracted from the Gross Tumor Volume (GTV) in 0.35T MRgRT scans of 26 locally advanced and borderline resectable PDAC patients treated with 50 Gy RT in 5 fractions. Feature ratios between first (F1) and last (F5) fraction scan were correlated with progression free survival (PFS). Feature stability was assessed through region of interest (ROI) perturbation. RESULTS: Linear normalization of image intensity to median kidney value showed improved reproducibility of feature quantification. Histogram skewness change during treatment showed significant association with PFS (p = 0.005, HR = 2.75), offering a potential predictive biomarker of RT response. Stability analyses revealed a wide distribution of feature sensitivities to ROI delineation and was able to identify features that were robust to variability in contouring. CONCLUSIONS: This study presents a proof-of-concept for the use of quantitative image analysis in MRgRT for treatment response prediction and providing an analysis pipeline that can be utilized in future MRgRT radiomic studies.

Nonthermal effects of radiofrequency-field exposure on calcium dynamics in stem cell-derived neuronal cells: elucidation of calcium pathways.

Intracellular Ca(2+) spikes trigger cell proliferation, differentiation and cytoskeletal reorganization. In addition to Ca(2+) spiking that can be initiated by a ligand binding to its receptor, exposure to electromagnetic stimuli has also been shown to alter Ca(2+) dynamics. Using neuronal cells differentiated from a mouse embryonic stem cell line and a custom-built, frequency-tunable applicator, we examined in real time the altered Ca(2+) dynamics and observed increases in the cytosolic Ca(2+) in response to nonthermal radiofrequency (RF)-radiation exposure of cells from 700 to 1100 MHz. While about 60% of control cells (not exposed to RF radiation) were observed to exhibit about five spontaneous Ca(2+) spikes per cell in 60 min, exposure of cells to an 800 MHz, 0.5 W/kg RF radiation, for example, significantly increased the number of Ca(2+) spikes to 15.7+/-0.8 (P<0.05). The increase in the Ca(2+) spiking activities was dependent on the frequency but not on the SAR between 0.5 to 5 W/kg. Using pharmacological agents, it was found that both the N-type Ca(2+) channels and phospholipase C enzymes appear to be involved in mediating increased Ca(2+) spiking. Interestingly, microfilament disruption also prevented the Ca(2+) spikes. Regulation of Ca(2+) dynamics by external physical stimulation such as RF radiation may provide a noninvasive and useful tool for modulating the Ca(2+)-dependent cellular and molecular activities of cells seeded in a 3D environment for which only a few techniques are currently available to influence the cells.

Modeling of carbon fiber couch attenuation properties with a commercial treatment planning system.

The purpose of this work is to evaluate the modeling of carbon fiber couch attenuation properties with a commercial treatment planning system (TPS, Pinnacle3, v8.0d). A carbon fiber couch (Brain-Lab) was incorporated into the TPS by automatic contouring of all transverse CT slices. The couch shape and dimensions were set according to the vendor specifications. The couch composition was realized by assigning appropriate densities to the delineated contours. The couch modeling by the TPS was validated by absolute dosimetric measurements. A phantom consisting of several solid water slabs was CT scanned, the CT data set was imported into the TPS, and the carbon fiber couch was auto-contoured. Open (unblocked) field plans for different gantry angles and field sizes were generated. The doses to a point at 3 cm depth, placed at the linac isocenter, were computed. The phantom was irradiated according to the dose calculation setup and doses were measured with an ion chamber. In addition, percent depth dose (PDD) curves were computed as well as measured with radiographic film. The calculated and measured doses, transmissions, and PDDs were cross-compared. Doses for several posterior fields (0 degree, 30 degrees, 50 degrees, 75 degrees, 83 degrees) were calculated for 6 and 18 MV photon beams. For model validation a nominal field size of 10 x 10 cm2 was chosen and 100 MU were delivered for each portal. The largest difference between computed and measured doses for those posterior fields was within 1.7%. A comparison between computed and measured transmissions for the aforementioned fields was performed and the results were found to agree within 1.1%. The differences between computed and measured doses for different field sizes, ranging from 5 x 5 cm2 to 25 x 25 cm2 in 5 cm increments, were within 2%. Measured and computed PDD curves with and without the couch agree from the surface up to 30 cm depth. The PDDs indicate a surface dose increase resulting from the carbon fiber couch field modification. The carbon fiber couch attenuation for individual posterior oblique fields (75 degrees) can be in excess of 8% depending on the beam energy and field size. When the couch is contoured in Pinnacle3 its attenuation properties are modeled to within 1.7% with respect to measurements. These results demonstrate that appropriate contouring together with relevant density information for the contours is sufficient for adequate modeling of carbon fiber supporting devices by modern commercial treatment planning systems.

Gene expression does not change significantly in C3H 10T(1/2) cells after exposure to 847.74 CDMA or 835.62 FDMA radiofrequency radiation.

In vitro experiments with C3H 10T(1/2) mouse cells were performed to determine whether Frequency Division Multiple Access (FDMA) or Code Division Multiple Access (CDMA) modulated radiofrequency (RF) radiations induce changes in gene expression. After the cells were exposed to either modulation for 24 h at a specific absorption rate (SAR) of 5 W/ kg, RNA was extracted from both exposed and sham-exposed cells for gene expression analysis. As a positive control, cells were exposed to 0.68 Gy of X rays and gene expression was evaluated 4 h after exposure. Gene expression was evaluated using the Affymetrix U74Av2 GeneChip to detect changes in mRNA levels. Each exposure condition was repeated three times. The GeneChip data were analyzed using a two-tailed t test, and the expected number of false positives was estimated from t tests on 20 permutations of the six sham RF-field-exposed samples. For the X-ray-treated samples, there were more than 90 probe sets with expression changes greater than 1.3-fold beyond the number of expected false positives. Approximately one-third of these genes had previously been reported in the literature as being responsive to radiation. In contrast, for both CDMA and FDMA radiation, the number of probe sets with an expression change greater than 1.3-fold was less than or equal to the expected number of false positives. Thus the 24-h exposures to FDMA or CDMA RF radiation at 5 W/kg had no statistically significant effect on gene expression.

Experience with a small animal hyperthermia ultrasound system (SAHUS): report on 83 tumours.

An external local ultrasound (US) system was developed to induce controlled hyperthermia of subcutaneously implanted tumours in small animals (e.g., mice and rats). It was designed to be compatible with a small animal positron emission tomography scanner (microPET) to facilitate studies of hyperthermia-induced tumour re-oxygenation using a PET radiopharmaceutical, but it is applicable for any small animal study requiring controlled heating. The system consists of an acrylic applicator bed with up to four independent 5 MHz planar disc US transducers of 1 cm in diameter, a four-channel radiofrequency (RF) generator, a multiple thermocouple thermometry unit, and a personal computer with custom monitoring and controlling software. Although the system presented here was developed to target tumours of up to 1 cm in diameter, the applicator design allows for different piezoelectric transducers to be exchanged and operated within the 3.5-6.5 MHz band to target different tumour sizes. Temperature feedback control software was developed on the basis of a proportional-integral-derivative (PID) approach when the measured temperatures were within a selectable temperature band about the target temperature. Outside this band, an on/off control action was applied. Perfused tissue-mimicking phantom experiments were performed to determine optimum controller gain constants, which were later employed successfully in animal experiments. The performance of the SAHUS (small animal hyperthermia ultrasound system) was tested using several tumour types grown in thighs of female nude (nu/nu) mice. To date, the system has successfully treated 83 tumours to target temperatures in the range of 41-43 degrees C for periods of 65 min on average.

Dose-mass inverse optimization for minimally moving thoracic lesions.

In the past decade, several different radiotherapy treatment plan evaluation and optimization schemes have been proposed as viable approaches, aiming for dose escalation or an increase of healthy tissue sparing. In particular, it has been argued that dose-mass plan evaluation and treatment plan optimization might be viable alternatives to the standard of care, which is realized through dose-volume evaluation and optimization. The purpose of this investigation is to apply dose-mass optimization to a cohort of lung cancer patients and compare the achievable healthy tissue sparing to that one achievable through dose-volume optimization. Fourteen non-small cell lung cancer (NSCLC) patient plans were studied retrospectively. The range of tumor motion was less than 0.5 cm and motion management in the treatment planning process was not considered. For each case, dose-volume (DV)-based and dose-mass (DM)-based optimization was performed. Nine-field step-and-shoot IMRT was used, with all of the optimization parameters kept the same between DV and DM optimizations. Commonly used dosimetric indices (DIs) such as dose to 1% the spinal cord volume, dose to 50% of the esophageal volume, and doses to 20 and 30% of healthy lung volumes were used for cross-comparison. Similarly, mass-based indices (MIs), such as doses to 20 and 30% of healthy lung masses, 1% of spinal cord mass, and 33% of heart mass, were also tallied. Statistical equivalence tests were performed to quantify the findings for the entire patient cohort. Both DV and DM plans for each case were normalized such that 95% of the planning target volume received the prescribed dose. DM optimization resulted in more organs at risk (OAR) sparing than DV optimization. The average sparing of cord, heart, and esophagus was 23, 4, and 6%, respectively. For the majority of the DIs, DM optimization resulted in lower lung doses. On average, the doses to 20 and 30% of healthy lung were lower by approximately 3 and 4%, whereas lung volumes receiving 2000 and 3000 cGy were lower by 3 and 2%, respectively. The behavior of MIs was very similar. The statistical analyses of the results again indicated better healthy anatomical structure sparing with DM optimization. The presented findings indicate that dose-mass-based optimization results in statistically significant OAR sparing as compared to dose-volume-based optimization for NSCLC. However, the sparing is case-dependent and it is not observed for all tallied dosimetric endpoints.

Simultaneous delivery of electron beam therapy and ultrasound hyperthermia using scanning reflectors: a feasibility study.

PURPOSE: The feasibility of simultaneously delivering external electron beam radiation and superficial hyperthermia using a scanning ultrasound reflector-array system (SURAS) was experimentally investigated and demonstrated. METHODS AND MATERIALS: A new system uses a scanning reflector to distribute the acoustic energy from a planar ultrasound array over the surface of the target volume. External photon/electron beams can be concurrently delivered with hyperthermia by irradiating through the scanning reflectors. That is, this system enables the acoustic waves and the radiation beams to enter the target volume from the same direction. Reflectors were constructed of air-equivalent materials for maximum acoustic reflection and minimum radiation attenuation. Acoustically, the air reflectors were compared to brass reflectors (assumed ideal) for reflectivity and specular quality using several single transducers ranging in frequency from 0.68 to 4.8 MHz. The relative reflectivity was determined from acoustic power measurements using a force-balance technique. The specular quality was assessed by comparing the acoustic pressure fields reflected by air reflectors with those reflected by brass reflectors. Also, acoustic pressure fields generated by a SURAS prototype for two different arrays (2.24 and 4.5 MHz) were measured to investigate field distribution variations as a function of the distance separating the array and the scanning reflector. All pressure fields were measured with a hydrophone in a degassed water tank. Finally, to determine the effect of the air reflectors on electron dose distributions, these were measured using film in a water-equivalent solid phantom after passage of a 20 MeV electron beam through the SURAS. These measurements were performed with the reflector scanning continuously across the electron beam and at rest within the electron beam. RESULTS: The measurements performed using single ultrasound transducers showed that the air reflectors had power reflectivities of 87-96% that of brass, and that for smooth surfaces the reflections from air reflectors were as specular as those from brass reflectors. Acoustic pressure fields measurements of the SURAS for two different arrays showed that the 50% pressure amplitude contours were well-distributed across the projected surface area of the array for different distances separating the array and the reflector. Finally, film dosimetry showed that the electron dose distribution was not affected by the air reflector of the SURAS either for the scanning case or the stationary case. This indicates that the reflectors as made are basically water-equivalent in terms of high energy ionizing radiation. The measured isodoses also indicate that the constructed SURAS prototype would allow the delivery of adequate radiation (90% isodose) to a depth of 2.0 cm. CONCLUSIONS: The results presented show that the SURAS design has the potential to deliver hyperthermia to large superficial tumors, while allowing simultaneous irradiation with 20 MeV electron beams without adverse effects on the radiation dose delivery.

High dose-rate induced temperature artifacts: thermometry considerations for simultaneous interstitial thermoradiotherapy.

PURPOSE: The goal of the present study was to investigate the effect of high dose-rate radiation on a flouroptic thermometry system commonly used during microwave hyperthermia. METHODS AND MATERIALS: Measurements were performed by placing the flouroptic thermometry sensors at distances of < or = 1.5, 5, 10, and 15 mm from a remote afterloading high dose-rate 192Ir source in a water bath (at two different temperatures) and in a tissue equivalent radiation bolus medium. A simulated volumetric clinical setup using a radiation bolus medium was performed with thermometry sensors placed at 1.5, 7.5, 8.4, and 10.6 mm from a scanning high dose-rate source. RESULTS: It was found that high dose-rate radiation caused thermometry artifacts greater than 1.5 degrees C within 2 min for flouroptic thermometers placed 1.5 mm from a 5 Ci activity high dose-rate source. Simple calculations showed that artifacts of this magnitude could not be due to any heating caused by the energy deposited by the high dose-rate source. The artifact decayed, but was still evident 24 h after the exposure. The effect strongly depended on distance with a 0.7 degrees C artifactual increase in temperature seen for the probe 5 mm from the high dose-rate source. Moreover, experiments performed under conditions that represented a clinical setup with a 7 Ci high dose-rate source showed that for exposure times of 10 s, at distances of 1.5 mm, significant artifacts (> 0.5 degrees C) are produced. CONCLUSIONS: These findings indicate that high dose-rate-induced temperature artifacts should be taken into account in the quality assurance procedures for the treatment of patients with simultaneous interstitial thermoradiotherapy.

An ultrasound system for simultaneous ultrasound hyperthermia and photon beam irradiation.

PURPOSE: An existing ultrasound system has been adapted for simultaneous use with external photon beam irradiation. The system is being used to investigate the potential for increased biological benefit of simultaneously combined hyperthermia and external beam irradiation with currently achievable temperature distributions. METHODS AND MATERIALS: An existing clinical ultrasound system has been modified for simultaneous operation with a 60Co teletherapy machine. The generator, thermometry system, computer, and applicators are located inside the treatment room, while the monitor and system control are located at the control console. Two approaches have been used clinically to combine the two modalities. In the first approach, an en-face setup is used in which the ultrasound beam and the photon beam travel through the same window of entry to the tumor. This is acheived by a reflecting system designed to deflect the ultrasound to the tumor while positioning the ultrasound transducer outside the radiation beam. The reflecting system consists of water and water-equivalent materials except for a 1 mm sheet of polished brass that is used as the reflector. The relative pressure fields were measured in water at the same distance from the ultrasound source using a scanning hydrophone with and without the reflector at the two operating frequencies of the device (1.0 and 3.4 MHz) for two applicators. Radiation dosimetry measurements were performed to determine the relationship between 60Co irradiation through the reflector and absorbed dose. In the second approach the ultrasound and the radiation beam travel into the tumor from different windows of entry such that the radiation beam passes through no portion of the water bolus prior to entering the patient. We have termed this approach the orthogonal approach. For both approaches, the radiation fraction is given in the middle of an uninterrupted 60-min hyperthermia treatment. RESULTS: The system modifications did not impair the ability to effectively deliver ultrasound hyperthermia or 60Co teletherapy. With the en-face approach the ultrasonic patterns generated with and without the reflector demonstrated that the ultrasound system maintained both a uniform and controllable heating pattern. The 60Co beam had no effect on the performance of the thermocouple thermometers. The radiation beam is attenuated nearly uniformly by the reflector system. To date, 10 patients have been treated with the en-face approach and 12 have been treated with the orthogonal approach (90 treatments). CONCLUSIONS: The clinical implementation of ultrasound hyperthermia simultaneous with 60Co irradiation is technically and clinically feasible without any complications or hazards to the patient. The implementation of a reflecting device allows en-face delivery of both the ultrasound and 60Co irradiation. Temperatures obtained during simultaneous treatments are comparable to those historically obtained during sequential treatments with the same commercial ultrasound device.

On the assumption of negligible heat diffusion during the thermal measurement of a nonuniform specific absorption rate.

The problem of measuring a spatially varying specific absorption rate (SAR) by thermal techniques is treated both analytically and numerically. It is shown that, unless the measurement is attempted at an inflection point of the SAR distribution, it will be confounded by thermal diffusion within the medium. Rules of thumb are provided to enable the experimenter to gauge the thermal conduction contributions (i.e. error) to the thermally determined SAR near a spatial extremum. The simplest of these is that the width t(m) [s] of the time window, over which temperature variation associated with SAR is measured, should satisfy the inequality t(m)

Radiofrequency electromagnetic fields do not alter the cell cycle progression of C3H 10T and U87MG cells.

The effects of exposure to radiofrequency electromagnetic fields (RF EMFs) on cell cycle progression of mouse fibroblasts C3H 10T(1/2) and human glioma U87MG cells were determined by the flow cytometric bromodeoxyuridine pulse-chase method. Cells were exposed to a frequency-modulated continuous wave at 835.62 MHz or a code division multiple access RF EMF centered on 847.74 MHz at an average specific absorption rate of 0.6 W/kg. Five cell cycle parameters, including the transit of cells through G(1), G(2) and S phase and the probability of cell division, were examined immediately after the cells were placed in the fields or after they had been kept in the fields for up to 100 h. The only significant change observed in the study was that associated with C3H 10T(1/2) cell cultures moving into plateau phase toward the later times in the long-exposure experiment. No changes in the cell cycle parameters were observed in cells exposed to either mode of RF EMFs when compared to sham-exposed cells in either of the cell lines studied during the entire experimental period. The results show that exposure to RF EMFs, at the frequencies and power tested, does not have any effect on cell progression in vitro.

Chromosome damage and micronucleus formation in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (847.74 MHz, CDMA).

Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m(2) (95 mW/cm(2)). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of gamma radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 +/- 0.3 degrees C. Immediately after the exposures, lymphocytes were cultured at 37 +/- 1 degrees C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of gamma-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.

Measurement of DNA damage after exposure to 2450 MHz electromagnetic radiation.

Recent reports suggest that exposure to 2450 MHz electromagnetic radiation causes DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in cells of rat brain irradiated in vivo (Lai and Singh, Bioelectromagnetics 16, 207-210, 1995; Int. J. Radiat. Biol. 69, 513-521, 1996). Therefore, we endeavored to determine if exposure of cultured mammalian cells in vitro to 2450 MHz radiation causes DNA damage. The alkaline comet assay (single-cell gel electrophoresis), which is reportedly the most sensitive method to assay DNA damage in individual cells, was used to measure DNA damage after in vitro 2450 MHz irradiation. Exponentially growing U87MG and C3H 10T1/2 cells were exposed to 2450 MHz continuous-wave (CW) radiation in specially designed radial transmission lines (RTLs) that provided relatively uniform microwave exposure. Specific absorption rates (SARs) were calculated to be 0.7 and 1.9 W/kg. Temperatures in the RTLs were measured in real time and were maintained at 37 +/- 0.3 degrees C. Every experiment included sham exposure(s) in an RTL. Cells were irradiated for 2 h, 2 h followed by a 4-h incubation at 37 degrees C in an incubator, 4 h and 24 h. After these treatments samples were subjected to the alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-267, 1992). Images of comets were digitized and analyzed using a PC-based image analysis system, and the "normalized comet moment" and "comet length" were determined. No significant differences were observed between the test group and the controls after exposure to 2450 MHz CW irradiation. Thus 2450 MHz irradiation does not appear to cause DNA damage in cultured mammalian cells under these exposure conditions as measured by this assay.

Measurement of DNA damage after exposure to electromagnetic radiation in the cellular phone communication frequency band (835.62 and 847.74 MHz).

Mouse C3H 10T1/2 fibroblasts and human glioblastoma U87MG cells were exposed to cellular phone communication frequency radiations to investigate whether such exposure produces DNA damage in in vitro cultures. Two types of frequency modulations were studied: frequency-modulated continuous-wave (FMCW), with a carrier frequency of 835.62 MHz, and code-division multiple-access (CDMA) centered on 847.74 MHz. Exponentially growing (U87MG and C3H 10T1/2 cells) and plateau-phase (C3H 10T1/2 cells) cultures were exposed to either FMCW or CDMA radiation for varying periods up to 24 h in specially designed radial transmission lines (RTLs) that provided relatively uniform exposure with a specific absorption rate (SAR) of 0.6 W/kg. Temperatures in the RTLs were monitored continuously and maintained at 37 +/- 0.3 degrees C. Sham exposure of cultures in an RTL (negative control) and 137Cs gamma-irradiated samples (positive control) were included with every experiment. The alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-269, 1992) was used to measure DNA damage. No significant differences were observed between the test group exposed to FMCW or CDMA radiation and the sham-treated negative controls. Our results indicate that exposure of cultured mammalian cells to cellular phone communication frequencies under these conditions at an SAR of 0.6 W/kg does not cause DNA damage as measured by the alkaline comet assay.

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro.

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.

Cytogenetic studies in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (835.62 MHz, FDMA).

Freshly collected peripheral blood samples from four healthy human volunteers were diluted with RPMI 1640 tissue culture medium and exposed in sterile T-75 tissue culture flasks in vitro for 24 h to 835.62 MHz radiofrequency (RF) radiation, a frequency employed for customer-to-base station transmission of cellular telephone communications. An analog signal was used, and the access technology was frequency division multiple access (FDMA, continuous wave). A nominal net forward power of 68 W was used, and the nominal power density at the center of the exposure flask was 860 W/m(2). The mean specific absorption rate in the exposure flask was 4.4 or 5.0 W/kg. Aliquots of diluted blood that were sham-exposed or exposed in vitro to an acute dose of 1.50 Gy of gamma radiation were used as negative or positive controls. Immediately after the exposures, the lymphocytes were stimulated with a mitogen, phytohemagglutinin, and cultured for 48 or 72 h to determine the extent of genetic damage, as assessed from the frequencies of chromosomal aberrations and micronuclei. The extent of alteration in the kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to mitotic indices, incidence of exchange aberrations, excess fragments, binucleate cells, and micronuclei. In contrast, the response of the lymphocytes exposed to gamma radiation was significantly different from both RF-radiation- and sham-exposed cells for all of these indices. Thus, under the experimental conditions tested, there is no evidence for the induction of chromosomal aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 835.62 MHz RF radiation at SARs of 4.4 or 5.0 W/kg.

Neoplastic transformation in C3H 10T(1/2) cells after exposure to 835.62 MHz FDMA and 847.74 MHz CDMA radiations.

The effect of radiofrequency (RF) radiation in the cellular phone communication range (835.62 MHz frequency division multiple access, FDMA; 847.74 MHz code division multiple access, CDMA) on neoplastic transformation frequency was measured using the in vitro C3H 10T(1/2) cell transformation assay system. To determine if 835.62 MHz FDMA or 847.74 MHz CDMA radiations have any genotoxic effects that induce neoplastic transformation, C3H 10T(1/2) cells were exposed at 37 degrees C to either of the above radiations [each at a specific absorption rate (SAR) of 0.6 W/kg] or sham-exposed at the same time for 7 days. After the culture medium was changed, the cultures were transferred to incubators and refed with fresh growth medium every 7 days. After 42 days, the cells were fixed and stained with Giemsa, and transformed foci were scored. To determine if exposure to 835.62 MHz FDMA or 847.74 MHz CDMA radiation has any epigenetic effects that can promote neoplastic transformation, cells were first exposed to 4.5 Gy of X rays to induce the transformation process and then exposed to the above radiations (SAR = 0.6 W/kg) in temperature-controlled irradiators with weekly refeeding for 42 days. After both the 7-day RF exposure and the 42-day RF exposure after X irradiation, no statistically significant differences in the transformation frequencies were observed between incubator controls, the sham-exposed (maintained in irradiators without power to the antenna), and the 835.62 MHz FDMA or 847.74 MHz CDMA-exposed groups.

Measurement of DNA damage in mammalian cells exposed in vitro to radiofrequency fields at SARs of 3-5 W/kg.

In the present study, we determined whether exposure of mammalian cells to 3.2-5.1 W/kg specific absorption rate (SAR) radiofrequency fields could induce DNA damage in murine C3H 10T(1/2) fibroblasts. Cell cultures were exposed to 847.74 MHz code-division multiple access (CDMA) and 835.62 frequency-division multiple access (FDMA) modulated radiations in radial transmission line (RTL) irradiators in which the temperature was regulated to 37.0 +/- 0.3 degrees C. Using the alkaline comet assay to measure DNA damage, we found no statistically significant differences in either comet moment or comet length between sham-exposed cells and those exposed for 2, 4 or 24 h to CDMA or FDMA radiations in either exponentially growing or plateau-phase cells. Further, a 4-h incubation after the 2-h exposure resulted in no significant changes in comet moment or comet length. Our results show that exposure of cultured C3H 10T(1/2) cells at 37 degrees C CDMA or FDMA at SAR values of up to 5.1 W/kg did not induce measurable DNA damage.

Proto-oncogene mRNA levels and activities of multiple transcription factors in C3H 10T 1/2 murine embryonic fibroblasts exposed to 835.62 and 847.74 MHz cellular phone communication frequency radiation.

This study was designed to determine whether two differently modulated radiofrequencies of the type generally used in cellular phone communications could elicit a general stress response in a biological system. The two modulations and frequencies studied were a frequency-modulated continuous wave (FMCW) with a carrier frequency of 835.62 MHz and a code division multiple-access (CDMA) modulation centered on 847.74 MHz. Changes in proto-oncogene expression, determined by measuring Fos, Jun, and Myc mRNA levels as well as by the DNA-binding activity of the AP1, AP2 and NF-kappaB transcription factors, were used as indicators of a general stress response. The effect of radiofrequency exposure on proto-oncogene expression was assessed (1) in exponentially growing C3H 10T 1/2 mouse embryo fibroblasts during their transition to plateau phase and (2) during transition of serum-deprived cells to the proliferation cycle after serum stimulation. Exposure of serum-deprived cells to 835.62 MHz FMCW or 847.74 MHz CDMA microwaves (at an average specific absorption rate, SAR, of 0.6 W/kg) did not significantly change the kinetics of proto-oncogene expression after serum stimulation. Similarly, these exposures did not affect either the Jun and Myc mRNA levels or the DNA-binding activity of AP1, AP2 and NF-kappaB in exponential cells during transit to plateau-phase growth. Therefore, these results suggest that the radiofrequency exposure is unlikely to elicit a general stress response in cells of this cell line under these conditions. However, statistically significant increases (approximately 2-fold, P = 0.001) in Fos mRNA levels were detected in exponential cells in transit to the plateau phase and in plateau-phase cells exposed to 835.62 MHz FMCW microwaves. For 847.74 MHz CDMA exposure, the increase was 1.4-fold (P = 0.04). This increase in Fos expression suggests that expression of specific genes could be affected by radiofrequency exposure.

Radiofrequency electromagnetic fields have no effect on the in vivo proliferation of the 9L brain tumor.

The intracranial 9L tumor model was used to determine if exposure to a radiofrequency (RF) electromagnetic field similar to those used in cellular telephone has any effects on the growth of a central nervous system tumor. Fischer 344 rats implanted with different numbers of 9L gliosarcoma cells were exposed to 835.62 MHz frequency-modulated continuous wave (FMCW) or 847.74 MHz code division multiple access (CDMA) RF field with nominal slot-average specific absorption rates in the brain of 0.75 +/- 0.25 W/kg. The animals were exposed to the RF field for 4 h a day, 5 days a week starting 4 weeks prior to and up to 150 days after the implantation of tumor cells. Among sham-exposed animals injected with 2 to 10 viable cells (group 1), the median survival was 70 days, with 27% of the animals surviving at 150 days. The median survival length and final survival fraction for animals injected with 11 to 36 viable cells (group 2) were 52 days and 14%, respectively, while the values for those injected with 37 to 100 cells (group 3) were 45 days and 0%. The animals exposed to CDMA or FMCW had similar survival parameters, and the statistical comparison of the survival curves for each of the groups 1, 2 and 3 showed no significant differences compared to sham-exposed controls.