Genotoxic Damage to Glioblastoma Cells Treated with 6 MV X-Radiation in The Presence or Absence of Methoxy Estradiol, IUDR or Topotecan.

OBJECTIVE: To explore the cumulative genotoxic damage to glioblastoma (GBM) cells, grown as multicellular spheroids, following exposure to 6 MV X-rays (2 Gy, 22 Gy) with or without, 2- methoxy estradiol (2ME2), iododeoxyuridine (IUDR) or topotecan (TPT), using the Picogreen assay. MATERIALS AND METHODS: The U87MG cells cultured as spheroids were treated with 6 MV X-ray using linear accelerator. Specimens were divided into five groups and irradiated using X-ray giving the dose of 2 Gy after sequentially incubated with one of the following three drug combinations: TPT, 2-ME2/TPT, IUDR/TPT or 2ME2/IUDR/ TPT. One specimen was used as the irradiated only sample (R). The last group was also irradiated with total dose of 22 Gy (each time 2 Gy) of 6 MV X-ray in 11 fractions and treated for three times. DNA damage was evaluated using the Picogreen method in the experimental study. RESULTS: R/TPT treated group had more DNA damage [double strand break (DSB)/single strand break (SSB)] compared with the untreated group (P<0.05). Moreover the R/ TPT group treated with 2ME2 followed by IUDR had maximum DNA damage in spheroid GBM indicating an augmented genotoxicity in the cells. The DNA damage was induced after seven fractionated irradiation and two sequential treatments with 2ME2/IUDR/TPT. To ensure accuracy of the slope of dose response curve the fractionated radiation was calculated as 7.36 Gy with respect to α/ß ratio based on biologically effective dose (BED) formulae. CONCLUSION: Cells treated with 2ME2/IUDR showed more sensitivity to radiation and accumulative DNA damage. DNA damage was significantly increased when GBM cells treated with TPT ceased at S phase due to the inhibition of topoisomerase enzyme and phosphorylation of Chk1 enzyme. These results suggest that R/TPT- treated cells increase sensitivity to 2ME2 and IUDR especially when they are used together. Therefore, due to an increase in the level of DNA damage (SSB vs. DSB) and impairment of DNA repair machinery, more cell death will occur. This in turn may improve the treatment of GBM.

DNA damage of glioblastoma multiform cells induced by Beta radiation of iodine-131 in the presence or absence of topotecan: a picogreen and colonogenic assay.

OBJECTIVE: Glioblastoma multiforme (GBM), one of the most common and aggressive malignant brain tumors, is highly resistant to radiotherapy. Numerous approaches have been pursued to find new radiosensitizers. We used a picogreen and colonogenic assay to appraise the DNA damage and cell death in a spheroid culture of GBM cells caused by iodine-131 (I-131) beta radiation in the presence of topotecan (TPT). MATERIALS AND METHODS: U87MG cells were cultured as spheroids with approximate diameters of 300 µm. Cells were treated with beta radiation of I-131 (at a dose of 2 Gy) and/ or TPT (1 µg/ml for 2 hours). The numbers of cells that survived were compared with untreated cells using a colonogenic assay. In addition, we evaluated possible DNA damages by the picogreen method. The relation between DNA damage and cell death was assessed in the experimental study of groups. RESULTS: The findings showed that survival fraction (SF) in the I-131+TPT group (39%) was considerably less than the I-131 group (58.92%; p<0.05). The number of single strand breaks (SSB) and double strand breaks (DSB), in the DNA of U87MG cells treated with beta radiation of I-131 and TPT (I-131+TPT) significantly increased compared to cells treated with only I-131 or TPT (p<0.05). The amount of SSB repair was more than DSB repair (p<0.05). The relationship between cell death and DNA damage was close (r≥0.6) and significant (p<0.05) in the irradiated and treated groups. Also the maximum rate of DNA repair occurred 24 hours after the treatments. A significant difference was not observed on other days of the restoration. CONCLUSION: The findings in the present study indicated that TPT can sensitize U87MG cells to radiation and increase DNA damages. Potentially, TPT can cause an increase in damage from DSB and SSB by its inhibitory effects on topoisomerase enzyme and the cell cycle. The increased complex damages following the use of a genotoxic agent and beta I-131 radiation, causes a significant increase the cell death because of the difficult repair process. By assessing the relationship between DNA damage and cell death, the picogreen method can be useful in predicting colonogenic assay. Consequently, it is suggested that co-treatment with I-131 beta radiation and TPT can improve GBM treatment.

DNA Damage Induced in Glioblastoma Cells by I-131: A Comparison between Experimental Data and Monte Carlo Simulation.

OBJECTIVE: The passage of ionizing radiation in living cells creates clusters of damaged nucleotides in DNA. In this study, DNA strand breaks induced by the beta particle of iodine-131 (I-131), have been determined experimentally and compared to Monte Carlo simulation results as a theoretical method of determining(131)I damage. MATERIALS AND METHODS: In this experimental study, in order to create single strand breaks (SSB) and double strand breaks (DSB) in the DNA, glioblastoma (GBM) cells were exposed to 10 mCi I-131, at a dose of 2 Gy. Damage of irradiated cells were evaluated quantitatively by the Fast Micromethod assay. The energy spectrum of electrons released in cells were obtained by the macroscopic Monte Carlo code (MCNP4c) and used as an input of the micro Monte Carlo code (MCDS). The percent of damage induced in cells was analyzed by Mann-Whitney test. RESULTS: A significant reduction (p<0.05) in fluorescence intensity in irradiated cells compared to control cells as determined by the Fast Micromethod assay represented induced SSB and DSB damages in the DNA of irradiated cells. Comparison of experimental and theoretical results showed that the difference between the percentages of SSB per Gy was about 7.4% and DSB was about 1% per Gy. CONCLUSION: The differences in experimental and theoretical results may be due to the algorithm of applied codes. Since the Fast Micromethod and other experimental techniques do not provide information about the amount of detailed and complex damages of DNA-like base damages, the applied Monte Carlo codes, due to their capability to predict the amount of detailed damages that occur in the DNA of irradiated cells, can be used in in vitro experiments and radiation protection areas.

Photoneutron and capture gamma dose equivalent for different room and maze layouts in radiation therapy.

In this paper the effect of treatment room and maze layout on the photoneutron and capture gamma dose equivalent in the maze was studied. MCNPX Monte Carlo (MC) code was used to simulate the Varian 2100 C/D Clinac 18 MV and four different room layouts. Two analytical methods, Wu-McGinley and McGinley, were used for dose calculations. The analytical methods overestimated the photoneutron dose (13-43 %) and gamma capture dose (16-95 %) comparing with the MC method at the maze entrance door. The results of MC method revealed that additional bend can cause a great reduction in photoneutron (5000 times) and capture gamma dose (50 times) in the maze entrance door.

Monte Carlo estimation of photoneutrons contamination from high-energy X-ray medical accelerators in treatment room and maze: a simplified model.

Despite all advantages associated with high-energy radiotherapy to improve therapeutic gain, the production of photoneutron via interaction of high-energy photons with high atomic number (Z) materials increases undesired dose to the patient and staff. Owing to the limitation and complication of experimental neutron dosimetry in mixed beam environment, including photon and neutron, the Monte Carlo (MC) simulation is a gold standard method for calculation of photoneutron contaminations. On the other hand, the complexity of treatment head makes the MC simulation more difficult and time-consuming. In this study, the possibility of using a simplified MC model for the simulation of treatment head has been investigated using MCNP4C general purpose MC code. As a part of comparative assessment strategy, the fluence, average energy and dose equivalent of photoneutrons were estimated and compared with other studies for several fields and energies at different points in treatment room and maze. The mean energy of photoneutrons was 0.17, 0.19 and 0.2 MeV at the patient plan for 10, 15 and 18 MeV, respectively. The calculated values differed, respectively, by a factor of 1.4, 0.7 and 0.61 compared with the reported measured data for 10, 15 and 18 MeV. Our simulation results in the maze showed that the neutron dose equivalent is attenuated by a factor of 10 for every 4.6 m of maze length while the related factor from Kersey analytical method is 5 m. The neutron dose equivalent was 4.1 mSv Gy(-1) at the isocentre and decreased to 0.79 mSv Gy(-1) at a distance of 100 cm away from the isocentre for 40 x 40 cm(2). There is good agreement between the data calculated using simplified model in this study and measurements. Considering the reported high uncertainties (up to 50%) in experimental neutron dosimetry, it can be concluded that the simplified model can be used as a useful tool for estimation of photoneutron contamination associated with high-energy photon radiotherapy.