Importance of Locations of Iron Ions to Elicit Cytotoxicity Induced by a Fenton-Type Reaction.

The impact of the site of the Fenton reaction, i.e., hydroxyl radical (•OH) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2), or pre-incubated with FeSO4 and then H2O2 was added after medium was replaced to remove iron ions or after the medium was not replaced. Cell lethality in rat thymocytes was estimated by measuring cell sizes using flow cytometry. High extracellular concentrations of FeSO4 exerted protective effects against H2O2-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO4 enhanced cell lethality induced by H2O2, whereas a pre-incubation with a high concentration of FeSO4 exerted protective effects. FeSO4 distributed extracellularly or on the surface of cells neutralized H2O2 outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of •OH, occurred inside cells. An assessment of plasmid DNA breakage showed that •OH induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated •OH does not appear to be DNA.

Preparation of an experimental mouse model lacking selenium-dependent glutathione peroxidase activities by feeding a selenium-deficient diet.

Relatively young (4-week-old) selenium deficient (SeD) mice, which lack the activity of selenium-dependent glutathione peroxidase (GSH-Px) isomers, were prepared using torula yeast-based SeD diet. Mice were fed the torula yeast-based SeD diet and ultra-pure water. Several different timings for starting the SeD diet were assessed. The weekly time course of liver comprehensive GSH-Px activity after weaning was monitored. Protein expression levels of GPx1 and 4 in the liver were measured by Western blot analysis. Gene expression levels of GPx1, 2, 3, 4, and 7 in the liver were measured by quantitative real-time PCR. Apoptotic activity of thymocytes after hydrogen peroxide (H2O2) exposure was compared. Thirty-day survival rates after whole-body X-ray irradiation were estimated. Pre-birth or right-after-birth starting of the SeD diet in dams was unable to lead to creation of SeD mice due to neonatal death. This suggests that Se is necessary for normal birth and healthy growing of mouse pups. Starting the mother on the SeD diet from 2 weeks after giving birth (SeD-trial-2w group) resulted in a usable SeD mouse model. The liver GSH-Px activity of the SeD-trial-2w group was almost none from 4 week olds, but the mice survived for more than 63 weeks. Protein and gene expression of GPx1 was suppressed in the SeD-trial-2w group, but that of GPx4 was not. The thymocytes of the SeD-trial-2w group were sensitive to H2O2-induced apoptosis. The SeD-trial-2w group was sensitive to whole-body X-ray irradiation compared with control mice. The SeD-trial-2w model may be a useful animal model for H2O2/hydroperoxide-induced oxidative stress.

Localized hydroxyl radical generation at mmol/L and mol/L levels in water by photon irradiation.

The generation of localized hydroxyl radical (•OH) in aqueous samples by low linear energy transfer irradiation was investigated. Several concentrations of 5,5-dimethyl-1-pyrroline-N-oxid solution (from 0.5 to 1,680 mmol/L) were prepared and irradiated with an identical dose of X-ray or γ-ray. The density of •OH generation in aqueous solution was evaluated by the electron paramagnetic resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxid as an electron paramagnetic resonance spin-trapping agent. The relationship between the molecular density of 5,5-dimethyl-1-pyrroline-N-oxid in the samples and the concentration of 5,5-dimethyl-1-pyrroline-N-oxid-OH generated in the irradiated samples was analyzed. Two different characteristic linear trends were observed in the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots, which suggested •OH generation in two fashions, i.e., mmol/L- and mol/L-level local concentrations. The dose, dose rate, and/or the energy of photon irradiation did not affect the shapes of the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots. Moreover, the addition of 5 mmol/L caffeine could cancel the contribution of mmol/L-level •OH generation, leaving a trace of mol/L-level •OH generation. Thus, the localized mmol/L- and mol/L-level generations of •OH, which were independent of experimental parameters such as dose, dose rate, and/or the energy of photon of low linear energy transfer radiation, were established.

Analysis of redox states of protic and aprotic solutions irradiated by low linear energy transfer carbon-ion beams using a 2,2-diphenyl-1-picrylhydrazyl radical.

The quantitative evaluation of changes in the redox state induced by low linear energy transfer (LET) radiations such as the plateau region of heavy-ion beams via formation of reactive oxygen species is of considerable importance to eliminate the adverse effects of radiation therapy on normal tissues adjacent to a tumour. In this study, a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) was used as a redox probe to estimate the redox states of protic and aprotic solutions irradiated by low LET carbon-ion (C-ion) beams. The dose dependence of the decrease in the absorption band due to DPPH˙ (which was solubilised by ß-cyclodextrin (ß-CD) in water) after irradiation with low LET C-ion beams (13 keV µm-1) was similar to that after X-irradiation. Similar results were obtained when H2O was replaced with methanol or acetonitrile although the slope values of the plots of the absorbance changes vs. radiation doses were twice larger as compared to the case in ß-CD-containing H2O. Moreover, DPPH˙ was more susceptible to the C-ion beam than to X-rays in isopropyl myristate (IPM), which is one of the saturated fatty acid esters.

Efficient protective activity of a planar catechin analogue against radiation-induced apoptosis in rat thymocytes.

About two thirds of biological damage due to low linear energy transfer (LET) radiation, such as X-rays and the plateau region of heavy-ion beams, is known to be caused by the hydroxyl radical (˙OH), the most powerful reactive oxygen species (ROS), generated via ionisation and excitation of water molecules. Thus, compounds having an efficient scavenging activity against ROS are expected to exhibit a radioprotective activity. A planar catechin analogue, where an isopropyl fragment was introduced into the catechol ring of (+)-catechin, showed an efficient protective effect against X-ray induced apoptosis in rat thymocytes compared to (+)-catechin. The planar catechin scavenged 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH˙) solubilised in water by ß-cyclodextrin about 10-fold faster than (+)-catechin in phosphate buffer (0.1 M, pH 7.4) at 298 K. Furthermore, the experimental log P value of the planar catechin (1.22) is reported to be significantly larger than that of (+)-catechin (0.44). The higher radical-scavenging activity and lipophilicity of the planar catechin than those of (+)-catechin may contribute in part to the higher protective activity against X-ray-induced apoptosis in rat thymocytes.

Synergistic Effects of Arsenite on Radiosensitization of Glioblastoma Cells.

BACKGROUND/AIM: Glioblastoma is a frequent type of brain tumor and is radioresistant. Arsenite, which crosses the blood-brain barrier, shows synergistic effects with radiation in vitro and in vivo. The mechanism remains unclear. MATERIALS AND METHODS: As synergistic radiosensitization has been reported in p53-deficient cancer cells, radiosensitization was evaluated using the glioblastoma cell line, U87MG-E6, which harbors inactivated p53, in comparison with the cell line, HCT116 p53 (-/-). Radiosensitivity was evaluated using clonogenic assays and detection of abnormal amplification of centrosomes (AAC). RESULTS: Synergistic effects of arsenite on radiosensitivity were observed in both cell lines. The radiosensitization induced by arsenite was abolished by N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger. Increased radiosensitivity by arsenite was also abolished following knock-down of BRCA2. In addition, the increased radiosensitization by arsenite was correlated with AAC, which was abolished by BRCA2 knock-down. CONCLUSION: We conclude that radiosensitization by arsenite is related to ROS and BRCA2 function.

Amplification of glutathione-mediated oxidative stress by catalase in an aqueous solution at hyperthermal temperatures.

The glutathione (GSH)-mediated superoxide (O2•-) generation in an aqueous solution and relation of hydrogen peroxide (H2O2) and effect of catalase were investigated. GSH-induced O2•- generation in hyperthermal temperatures was measured by the nitroblue tetrazolium (NBT) mehod. Heating an aqueous solution containing GSH caused superoxide from dissolved O2. H2O2 was generated simultaneously in this reaction mixture probably from the hydroperoxy radical (HO2•), which is equilibrated with O2•- in an aqueous condition, and then H2O2 consumed O2•-. Coexisting catalase in the reaction mixture, as a result, could increase O2•- generation. The catalase-exaggerated extracellular O2•- generation could give a harmful effect to living cells. This GSH-induced oxidative stress can be a part of mechanisms of hyperthermia therapy.

Non-invasive measurement of melanin-derived radicals in living mouse tail using X-band EPR.

The aim of this experiment is to measure in vivo generation of melanin-derived radicals non-invasively, as a quantifiable index of radio-biological effect. Melanin-derived radicals in a living intact mouse tail tip were non-invasively measured in very simple way using an X-band electron paramagnetic resonance spectrometer. Colored mouse strains, C57BL/6NCr, BDF1, and C3H/He, have clear EPR signal corresponding to melanin-derived radicals in the tail tip; however, albino mouse strains, BALB/cCr, ddY, ICR, have no EPR signals. An X-ray fraction of 2 Gy/day (1 Gy/min) was repeatedly irradiated to a C3H/He mouse tail skin every Monday to Friday for 4 weeks. In comparison to before starting irradiation, the C3H/He mouse tail skin became darker, like a suntan. The melanin-derived radicals in C3H/He mouse tail skin were increased in association with X-ray fractions. Melanin-derived radicals in mouse tail skin can be readily and chronologically measurable by using X-band EPR spectrometer, and can be a marker for a radiobiological effect in the skin.

A facile and rapid access to resveratrol derivatives and their radioprotective activity.

A facile and rapid access to resveratrol derivatives has been achieved based on palladium-catalyzed oxidative Heck reaction of aryl boronic acids with styrenes followed by demethylation in moderate to good yields. A series of resveratrol derivatives with various functional groups has been synthesized easily. The radioprotective activity of synthesized compounds has also been evaluated using rat thymocytes. The results revealed that some resveratrol derivatives efficiently protected the thymocytes from radiation-induced apoptosis.

Analysis of the antioxidative function of the radioprotective Japanese traditional (Kampo) medicine, hangeshashinto, in an aqueous phase.

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy- and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study was to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (•OH)-scavenging ability and the reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin-trapping method. The superoxide (O(2) (•-))-scavenging ability was estimated by an EPR redox probing method. Water suspensions of powdered HST and of its seven constitutive crude drugs were tested. In addition, some of the main water-soluble ingredients of the crude drugs were also tested. HST was found to scavenge both •OH and O(2) (•-). Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso) and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good •OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water-soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function in maintaining catalytic redox action. The antioxidative function of HST probably worked via a balance of scavenging ROS, reducing stable free radicals, and some minor oxidizing activities.

Arsenite induces premature senescence via p53/p21 pathway as a result of DNA damage in human malignant glioblastoma cells.

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced γH2AX foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated ß-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage.

High-throughput screening of radioprotectors using rat thymocytes.

We invented a high-throughput screening method for the examination of radioprotective activity of chemical compounds using rat thymocytes. X-irradiation of the rat thymocytes induced apoptosis, leading to a significant cell shrinkage, which could be easily detected and directly quantified by the flow cytometry analysis. The protective effect of some natural antioxidants against radiation induced apoptosis in the rat thymocytes, as well as their toxicities without X-irradiation, was successfully evaluated using this method. This method provides a powerful tool to develop novel radioprotectors without toxicity and can also be widely used to estimate other oxidative stress except for radiation.

Effectiveness of combined treatment using X-rays and a phosphoinositide 3-kinase inhibitor, ZSTK474, on proliferation of HeLa cells in vitro and in vivo.

ZSTK474 is a novel orally applicable phosphoinositide 3-kinase-specific inhibitor that strongly inhibits cancer cell proliferation. To further explore the antitumor effect of ZSTK474 for future clinical usage, we studied its combined effects with radiation. The proliferation of HeLa cells was inhibited by treatment with X-rays alone or ZSTK474 alone. Combination treatment using X-rays then ZSTK474 given orally for 8 days, starting 24 h post-irradiation, significantly enhanced cell growth inhibition. The combined effect was also observed for clonogenic survival with continuous ZSTK474 treatment. Western blot analysis showed enhanced phosphorylation of Akt and GSK-3ß by X-irradiation, whereas phosphorylation was inhibited by ZSTK474 treatment alone. Treatment with ZSTK474 after X-irradiation also inhibited phosphorylation, and remarkably inhibited xenograft tumor growth. Combined treatment with X-rays and ZSTK474 has greater therapeutic potential than radiation or drug therapy alone, both in vitro and in vivo.

Chemopreventive agent sulforaphane enhances radiosensitivity in human tumor cells.

Sulforaphane (SFN), an isothiocyanate derived from broccoli and other cruciferous vegetables, is a positive regulator of phase II detoxification enzymes and is highly effective in protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN also enhances radiosensitivity in human tumor cells. Cell survival in HeLa human cervix carcinoma cells pretreated with SFN was significantly lower than in cells treated with radiation only. Constant-field gel electrophoresis and a gamma-H2AX foci assay showed marked inhibition of DSB repair in irradiated cells treated with SFN, while little inhibition was observed in cells with DMSO (control). In addition, immunofluorescence experiments revealed a significant delay in Rad51 (a key protein for homologous recombination repair) foci formation and disappearance in irradiated cells treated with SFN when compared to the cells with X-irradiation alone. The dephosphorylation of DNA-PKcs (a critical nonhomologous end joining protein) was also markedly delayed by SFN pretreatment in irradiated cells. These DSB repair inhibition data partially support the high apoptotic frequency of irradiated cells pretreated with SFN. Furthermore, the combined treatment of X-rays and SFN (i.p. 300 micromol/kg) in the xenograft model with HeLa cells showed efficient inhibition of in vivo tumor growth. To the best of our knowledge, our study is the first report showing SFN-enhanced radiosensitivity of tumor cells in vitro and in vivo, which opens the door for a multitude of clinical applications for chemoradiotherapy using SFN.

Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells.

Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and gamma-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.