We investigated the role of the caspase activation cascade in apoptosis induced by ionizing radiation or hydrogen peroxide (H₂O₂) in human leukemia HL60 cells. Electron paramagnetic resonance (EPR) spectra revealed that hydroxyl and hydrogen radicals were generated in the culture medium after exposure to radiation or H₂O₂. Initial accumulation of DNA fragments at 2 h after exposure was delayed in irradiated cells compared with H₂O₂-treated cells, although formation of abasic sites immediately after exposure was significantly higher in irradiated cells and similar quantities of hydroxyl radicals were produced under both conditions. Activity assay of caspases revealed that caspase-3, -8 and -9 were activated 2 h after exposure to H₂O₂, whereas in irradiated cells caspase-3 and -9 activation occurred 4 h after exposure but increased caspase-8 activation was not observed. Release of cytochrome c into cytosol was seen at 2 h after radiation and H₂O₂ treatment. Radiation did not affect proapoptotic proteins (Bax and Bid), whereas H ₂O₂ increased accumulation of Bax in the mitochondrial membrane 2 h to 6 h after treatment, independently of the truncation of Bid by activated caspase-8. Moreover, treatment with the caspase-8 inhibitor Z-IETD-FMK increased cell survival and prevented accumulation of DNA fragments in H₂O₂-treated cells, but not in irradiated cells. These results suggest that, unlike the caspase cascade of H₂O₂-induced apoptosis, cytochrome c and caspase-9 are important for the intrinsic pathway of radiation-induced apoptosis, independent of caspase-8.

View full abstract

Peroxiredoxin I (Prx-I), a key member of the peroxiredoxin family, reduces peroxides and equivalents through the thioredoxin system. Our previous work has shown that expression of Prx-I in mammalian cells increases following ionizing radiation (IR), indicating that Prx-I actively responds to IR-induced reactive oxygen species (ROS) and suggesting that Prx-I plays an important role in protecting cells from IR-induced death. To test this hypothesis, we suppressed the expression of Prx-I in SW480 cells by RNA interference. Our results show that IR induces the expression of Prx-I in SW480 cells in a dose- and time-dependent manner. The recombinant siRNA vector targeting Prx-I dramatically reduced the expression of Prx-I in SW480 cells. When Prx-I was knocked down in SW480 cells, the cells exhibited a decreased growth rate, a reduced antioxidant capability following IR and became more sensitive to IR-induced apoptosis. Together, our results demonstrate that Prx-I plays an important role in protecting cells from IR-induced cell death, which might be through scavenging IR-induced ROS in the cells.

View full abstract

The effect of magnetic field (MF) exposure on melatonin production was studied in female CD₂F₁(BALB/c x DBA/2) mice. The mice were exposed to a 50 Hz MF at 100 μT for 52 days and nocturnal urine was collected 1, 3, 7, 14, 16 and 23 days after the beginning of MF exposure. The animal room was illuminated for 12 h daily at 200 lux. To study the circadian rhythm of melatonin production, night and day samples of urine were collected once, at about 40 days after the beginning of MF exposure. Urinary 6-hydroxy melatonin sulfate (6-OHMS) was determined to assess melatonin production. The pineal glands were analyzed for melatonin content at the middle of the dark period. No statistically significant peak of melatonin was observed in either group. The light-regulated natural melatonin rhythm was absent in sham-exposed mice. The MF exposure caused a significant day-night difference in the 6-OHMS levels, but did not affect the total excretion of 6-OHMS during the 24-hour period. A possible interpretation of the findings is that MF exposure increases the sensitivity of the pineal gland to light in this strain normally insensitive to the circadian light variations. Further studies on interaction of light and MF exposure might help in understanding the inconsistencies of earlier research on MFs and melatonin.

View full abstract

The formation of products resulting from the O-glycoside bond cleavage following radiolysis of aqueous solutions of methyl-α-D-glucopyranoside (I), 3-O-methyl-α-D-glucopyranose (II), maltose, lactose, gentiobiose and cellobiose were studied. Radiation-induced destruction yields were also determined for dextran, laminarin and trimethylcelulose upon irradiation of their aqueous solutions. Oxygen, quinones and compounds capable of forming quinoid structures were found to inhibit radiation-induced homolytic destruction processes taking place in glycosides, di- and polysaccharides. The data obtained in this study enabled the authors to demonstrate an important role played by the fragmentation reaction of C-2 radicals which were generated from the starting substances in the formation of final radiolysis products.

View full abstract

We studied the relation between initial DNA double-strand breaks (DNA-DSB) and the rejoining kinetics of the strand breaks, as well as the OER (oxygen enhancement ratio) after low- and high-LET (linear energy transfer) radiations. CHO cells were exposed to 200 kVp X-rays or 80 keV/μm carbon ions under oxic and hypoxic conditions. DNA-DSB in the cells were analyzed by a static-field gel electrophoresis (SFGE). The kinetics of the rejoining could be described by a sum of fast and slow components. The initial released DNA after X-ray irradiation was higher for cells irradiated under an oxic condition than that under a hypoxic condition. The OER of DNA-DSB after X-ray irradiation was 5.7. This value decreased rapidly to be 3.4 with the fast component by 15 minutes. On the other hand, the OER of DNA-DSB after carbon ion irradiation was 2.2, and this value was not changed by rejoining incubation. The OER values for cell killing were 2.8 and 1.8 after X-ray and carbon ion irradiations, respectively. These values matched to the OER for DNA-DSB with complete rejoining. We conclude that the rejoining of DNA-DSB is an important factor in the mechanism of the oxygen effect.

View full abstract

The yield of OH radical induced by ionizing radiation was estimated by an empirical model; a prescribed diffusion model for a spur of single size applying to neutral water. Two representative spur distances were introduced, one for an incident primary charged particle and one for a representative secondary electron, to calculate chemical yields among active species in a spur. The total yield from the track was a combination of these primary and secondary yields. Two coefficients of this combination were the parameters of the present model. Based on an optimization of these parameters by existing experimental Fricke G-values, the present model estimates the yields of OH at the microsecond timescale after an irradiation, in a unified manner from electrons to heavy ions. The predicted yields of OH around the nanosecond timescale after an irradiation may be a relevant basis for a study on the mechanisms of radiation effects. This prediction by the present model was exemplified for electrons, photons and heavy ions (proton, helium, carbon, neon, argon and iron).

View full abstract

The reason why RBE for cell killing fell to less than unity (1.0) with very high-LET heavy-ions (⁴⁰Ar: 1,640 keV/μm; ⁵⁶Fe: 780, 1,200, 2,000 keV/μm) was explored by evaluating the fraction of non-hit cell (time-lapse observation) and cells undergoing interphase death (calculation based on our previous data). CHO cells were exposed to 4 Gy (30% survival dose) of Ar (1,640 keV/μm) or Fe-ions (2,000 keV/μm). About 20% of all cells were judged to be non-hit, and about 10% cells survived radiation damage. About 70% cells died after dividing at least once (reproductive death) or without dividing (interphase death). RBE for reproductive (RBE[R]) and interphase (RBE[I]) death showed a similar LET dependence with maximum around 200 keV/μm. In this LET region, at 30% survival level, about 10% non-survivors underwent interphase death. The corresponding value for very high-LET Fe-ions (2,000 keV/μm) was not particularly high (~15%), whereas that for X-rays was less than 3%. However, reproductive death (67%) predominated over interphase death (33%) even in regard to rather severely damaged cells (1% survival level) after exposure to Fe-ions (2,000 keV/μm ). These indicate that interphase death is a type of cell death characteristic for the cells exposed to high-LET radiation and is not caused by `cellular over kill effect'. Both NHF37 (non-hit fraction at 37% survival) and inactivation cross-section for reproductive death (σ[R]) began to increase when LET exceeded 100 keV/μm. The exclusion of non-hit fraction in the calculation of surviving fraction partially prevented the fall of RBE[R] when LET exceeded 200 keV/μm. On the other hand, the mean number of lethal damage per unit dose (NLD/Gy) showed the same LET-dependent pattern as RBE[R]. These suggest that the increase in non-hit fraction and σ[R] with an increasing LET is caused by enhanced clustering of ionization and DNA damage which lowers the energy efficiency for producing damage and RBE.

View full abstract

This study examined whether 2450 MHz continuous wave high frequency electromagnetic fields (HFEMF) could induce cancer-like changes in mouse C3H10T1/2 cells, and whether HFEMF could initiate malignant or synergistic transformation. Transformed foci, Type II and Type III, were independently counted as the experiment endpoint. The cells were exposed to HFEMF alone at a wide range of specific absorption rates (SARs) of 5 to 200 W/kg for 2 h and/or were treated with a known initiating chemical, methylcholanthrene (MC) (2.5 μg/ml). No significant differences were observed in the malignant transformation (Type II + Type III) frequency between the controls and HFEMF with or without 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.5 ng/ml), a tumor promoter that could enhance transformation frequency initiated by MC in multistage carcinogenesis. However, the transformation frequency for HFEMF at SAR of more than 100 W/kg with MC or MC plus TPA was increased compared with MC alone or MC plus TPA. On the other hand, the corresponding heat groups (heat alone, heat + MC, and heat + MC + TPA) did not increase transformation compared with each control level in C3H10T1/2 cells. This result suggests that 2450 MHz HFEMF could not contribute to the initiation stage of tumor formation, but it may contribute to the promotion stage at the extremely high SAR (100 W/kg).

View full abstract

We have investigated the radiosensitizing effect of doranidazole, a hypoxic cells radiosensitizer, using SCCVII tumor cells of C3H mice and CFPAC-1 and MIA PaCa-2 human pancreatic tumor cells. The radiosensitivity of hypoxic SCCVII cells in vitro increased with 1 mM doranidazole by a factor of 1.34 and 1.68, when determined by clonogenic survival and micronucleus (MN) formation, respectively. The radiation-induced growth delay of SCCVII tumors was significantly enhanced and the TCD_50/120 was reduced by a factor of 1.33 when 200 mg/kg doranidazole was injected, i.v., 20 min prior to tumor irradiation. The in vivo-in vitro excision assay showed that radiosensitivity of SCCVII cells in vivo increased by a factor of 1.47 with 200 mg/kg doranidazole. The radiation-induced growth delay of CFPAC-1 xenografts in nude mice was significantly enhanced and the TCD_50/90 was reduced by a factor of 1.30 by 200 mg/kg doranidazole. On the other hand, 200 mg/kg of doranidazole exerted no influence on the radiation-induced growth delay in MIA PaCa-2 xenografts. The tumor oxygenation status, as determined with an oxygen sensitive needle probe and the immunohistological study using pimonidazole, indicated that MIA PaCa-2 tumors are better oxygenated than CFPAC-1 tumors. The relatively well-oxygenated status in MIA PaCa-2 tumor may account for the lack of radiosensitization by doranidazole. It is concluded that the magnitude of radiosensitization of tumors by doranidazole is dependent on the oxygenation status of the tumors and that doranidazole may be useful in increasing the response of hypoxic human pancreatic tumor to IORT.

View full abstract