Hiroshi Maezawa

Design of a bioreductively-activated fluorescent pH probe for tumor hypoxia imaging

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wavelength absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts. UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with reduced background signal.

Synchrotron system for monochromatic uv irradiation (>140 nm) of biological material

An irradiation system of monochromatic uv radiation down to the wavelength of 140 nm was constructed for biological irradiation experiments in the vacuum-uv range using synchrotron radiation (SR) from the electron storage ring. The system consists of premirror chamber, vacuum-uv monochromator, irradiation chamber, and vacuum systems. Along with the detailed description of all components of the system, the installation at the storage ring and the performance characteristics are presented.

Bystander Cell Killing in Normal Human Fibroblasts is Induced by Synchrotron X-Ray Microbeams

Abstract The radiation-induced bystander response is defined as a response in cells that have not been directly targeted by radiation but that are in the neighborhood of cells that have been directly exposed. In the work described here, it is shown that bystander cell killing of normal human fibroblast WI-38 cells was induced by synchrotron microbeam X radiation. Cell nuclei in confluent WI-38 cells were irradiated with the microbeam. All of the cells on the dish were harvested and plated 24 h after irradiation. It was found that the bystander cell killing effect showed a parabolic relationship to the radiation dose when five cells were irradiated. At doses above 1.9 Gy, the surviving fraction increased to approximately 1.0. This suggests that induction of bystander cell killing may require some type of activity in the targeted cells, because the dose resulting in 37% cell survival was about 2.0 Gy. Bystander cell killing was suppressed by a pretreatment with aminoguanidine [an inhibitor of inducible nitric oxide (NO) synthase] or carboxy-PTIO (a scavenger of NO). These results suggest that NO is the chief initiator/mediator of bystander cell killing induced by X-ray microbeams.

Action spectra for inactivation of dry phage T1 after monochromatic (150-254 nm) synchrotron irradiation in the presence and absence of photoreactivation and dark repair.

Dry phage T1 was irradiated with monochromatic uv radiation (150-254 nm) in vacuo. The inactivation sensitivity was highest at 150 nm. On Escherichia coli Bs-1, the phage inactivation sensitivity was two and four times higher at 150 nm than at 254 and 230 nm, respectively. Action spectra for phage inactivation on both E. coli B and Bs-1 fit the absorption spectrum of phage DNA, except around 190 nm. The host-cell- reactivable fraction for vacuum-uv radiation (below 190 nm) was smaller than that with far-uv radiation. There was almost no photoreactivation at 150 nm, in contrast to a photoreactivation sector of about 0.3 at 254 nm.

Synthesis and photophysical properties of new SNARF derivatives as dual emission pH sensors

We report the synthesis and properties of two new seminaphthorhodafluor (SNARF) derivatives, SNARF-F and SNARF-Cl. Both these derivatives exhibit typical red shifts of absorption and fluorescence, and higher cell permeability as compared to traditional SNARF, while the pH-dependent dual-emission characteristics are well retained. In particular, the lower pK(a) (7.38) of SNARF-F makes it more suitable than traditional SNARF derivatives for intracellular applications.

Selection of endogenous 13C substrates for observation of intracellular metabolism using the dynamic nuclear polarization technique

PurposeThe aim of this study was to select a suitable substrate candidate for dynamic nuclear polarization (DNP) studies and demonstrate its utility for evaluating intracellular metabolism.Materials and methodsHyperpolarized substances included 1-13C-pyruvate (Pyr), 1-13C-glucose (Glc), and 1-13C-acetate. A DNP polarizer and a 600-MHz vertical small-bore scanner were used for 13C-MR spectroscopic measurements. After polarization for 1 h, the dissolved solution was injected via a capillary line into the nuclear magnetic resonance tube in the scanner. The sequential spectra of the hyperpolarized 13C-labeled substrates were acquired in durations of more than 120 s, and a thermal spectrum was obtained more than 1 h thereafter. FM3A cancer cells of mammary tumors were cultured for intracellular detection of the hyperpolarized 13C-substances.ResultsThe greatest sensitivity was found using Pyr with the longest T1 decay (51.5 s); and remarkably, the least sensitivity was observed using Glc with a signal decay of less than 2 s. An effective increase in sensitivity was shown using the other substances. The hyperpolarized intracellular study using 13C-Pyr showed distinct elevation of lactate levels.ConclusionThe DNP technique is useful for evaluating intracellular metabolism. However, Glc is not suitable for use with the DNP technique.

Development of photon microbeam irradiation system for radiobiology

Abstract In order to evaluate the risk of low dose or low-dose rate irradiation, a microbeam irradiation system using synchrotron radiation has been proposed, which overcomes the difficulty of the Poisson distribution of hit cells in the majority of the non-hit cell population. Up to now, we succeeded in developing a system having the following specifications. The smallest X-ray beam size obtained is 2 μm, practically 10 μm with X-ray energy of 5.35 keV. Cells grown and irradiated on specially designed dishes can be identified, after various treatments, for detection of irradiated response. Presently, the system works only in “point and shoot” mode. Automatic recognition and irradiation of cells, which enables the irradiation of 1000 cells/h, will soon be operative.

Iron(II) sulphate (Fricke solution) oxidation yields for 8.9 and 13.6 keV X-rays from synchrotron radiation

The oxidation yields (G) for 8.86 and 13.55 keV X-rays produced by synchrotron radiation were measured using an iron(II) sulphate (Fricke) solution. Monoenergetic X-rays were produced using a silicon crystal monochromator. The X-rays were absorbed in 0.4 M sulphuric acid-iron(II) sulphate solution and FeIII ion yields were measured and corrected for escape fractions resulting from scattering using Monte Carlo calculations. Doses in the solution were determined using a thin window, parallel plate chamber calibrated against a primary standard free-air chamber at the Electrotechnical Laboratory (Osaka, Japan). Yields (G) of 1.50 +/- 0.06 and 1.43 +/- 0.06 mumol J-1 were obtained for 8.86 and 13.55 keV X-rays respectively.

Lethal Effect of K-Shell Absorption of Intracellular Phosphorus on Wild-Type and Radiation Sensitive Mutants of Escherichia Coli

The present study was conducted to clarify the lethality of Auger cascades induced by the K-shell photoabsorption of phosphorus in Escherichia coli. Killing of wild-type and radiation-sensitive mutants of E. coli was examined. Three x-ray energies were chosen for irradiation; at 2.153 keV: the resonance peak of K-shell photoabsorption of phosphorus; at 2.146 and 2.160 keV: off-peak. Enhancement ratio, which was defined as the ratio of the killing sensitivity of 2.153 keV to that at 2.146 keV, were 1.32 to 1.54 for examined strains. Increment of absorbed energy calculated in entire cells for 2.153 keV radiation could not explain the degree of observed enhancement of killing. Lethality of Auger cascades depended on the killing sensitivity with x-rays which did not induce Auger cascades. The lethality for wild-type was lower than that for recombination repair-deficient mutants. It was concluded that one part of damages produced by Auger cascades was repaired in wild-type strains.

Radiosensitivity of mouse skin epithelial cell line established in serum-free culture: An alternative to animal use

A cultured line of murine skin epithelial cells was established to investigate the potential use of cultured cells as an alternative to animal use in radiation research. C3Hf/Sed newborn mouse skin cells have been successfully cultured in serum- and Ca(2+)-free medium with no terminal differentiation to keratinized cells. Presently, more than 25 passages have passed with no loss of stem cell capability. The radiosensitivity and repair of sublethal and potentially lethal radiation damages were investigated in this epithelial cell line. The population cell doubling time was 25 +/- 2.9 hr at 37 degrees C. The clonal growth of epithelial cells after irradiation was performed in the serum-free medium in the presence of lethally irradiated skin fibroblasts. Single dose survival curves of exponentially growing epithelial cells were investigated from the seventh to the twenty-third passages, and no significant changes in radiosensitivity and doubling time were found. The confluent epithelial cells also showed an identical sensitivity to radiation. The alpha/beta ratios of survival curves fitted by the linear quadratic model were 6.1 +/- 1.0 and 5.9 +/- 1.3 Gy for cells in exponential and confluent phases, respectively. The survival curve of epithelial cells left in confluence for 8 hr after irradiation showed a smaller beta value than that of cells plated immediately after irradiation with a resultant alpha/beta ratio of 9.5 +/- 3.8 Gy. This alpha/beta ratio was identical to those found in many animal experiments, suggesting a potential use of this cell line as an alternative to animal use. The magnitude of repair of sublethal damage following 6 Gy was greater than that following 3.9 Gy. Survival curves were also obtained following twice-a-day irradiations with no sign of rapid repopulation. These results are discussed by comparing with published in vivo and in vitro data.