Giichi Yoshii

Damage in DNA Irradiated with 1.2 MHz Ultrasound and Its Effect on Template Activity of DNA for RNA Synthesis

When aqueous DNA solution was irradiated with 1.2 MHz continuous ultrasound in the presence of cysteamine, the number of ultrasound-induced double-strand breaks of DNA was not influenced, but the number of ultrasound-induced single-strand breaks of DNA was reduced to about one-fifth that of the irradiated control. When the effect of cysteamine on the template activity of the ultrasound-irradiated DNA was investigated, the cysteamine was found to exert a leveling effect on the linear decrease of the template activity against ultrasonic intensity. Since cysteamine was known as an effective radical scavenger, the results of the experiment were regarded to suggest that (1) the double-strand breaks were exclusively induced by the mechanical effect of ultrasound, (2) the majority of single-strand breaks were produced by water radicals arising from cavitation, (3) the initial part in the decrease of the template activity was due to the double-strand breaks arising from mechanical effect, and (4) the further decrease of the template activity depended mainly on the single-strand breaks arising from water radicals.

Effect of intensity of 1.2 MHz ultrasound on change in DNA synthesis of irradiated mouse L cells

Cultured mouse L cells attached to circular plates were exposed to continuous wave 1.2 MHz ultrasound and the amount of 14C-thymidine (TdR) incorporated into cellular DNA was measured. Enhanced 14C-TdR incorporation per cell in attached cells was observed at ultrasonic intensities above 0.5, ISATA (0.8, ISPTA) W/cm2, although the total amount of incorporated 14C-TdR per sample decreased with increasing exposure energy at intensities above 1.5 (2.8) W/cm2. The presence of cavitation was evidenced by three kinds of physicochemical dosimeters; the liberation of iodine from KI-starch solution, a decrease in DNA molecular weight and sucrose hydrolysis. Collapse cavitation was detected at intensities above 1.5 (2.8) W/cm2 and stable cavitation was observed at 0.5 (0.8) W/cm2. These results suggest that the change in DNA synthesis of irradiated mouse L cells takes place independently of collapse cavitation, and may be due to stable cavitation.

The effect of γ-irradiation on deoxyribonucleoprotein as a template for RNA synthesis

Abstract 1. The effect of ionizing radiation on calf thymus DNA and deoxyribonucleoprotein which served as a template for RNA synthesis was investigated in the absence or presence of Ca 2+ . 2. In the absence of Ca 2+ , the template activity of calf thymus deoxyribonucleoprotein was decreased by γ-irradiation at a dose lower than 5 krads, whereas the template activity increased with a dose range from 10 to 30 krads and decreased again at a dose higher than 30 krads. On the other hand, in the presence of Ca 2+ , the increase of the template activity by γ-irradiation occurred at much lower dose than in the absence of Ca 2+ . 3. When Ca 2+ was added to the DNA solution, no change of the dose—effect curve of the template activity could be observed, compared with pure DNA. 4. These results are consistent with the interpretation that the increase of the template activity of deoxyribonucleoprotein is caused by the labilization of deoxyribonucleoprotein into the DNA and the protein on irradiation.

Reaction of the Hydrated Electron with Histone H1 and Related Compounds Studied by e.s.r. and Spin-trapping

The reactions of the hydrated electron with histone H1, protamine and related compounds (poly-L-lysine, poly-L-arginine and poly-D,L-alanine) were investigated by the spin-trapping technique. In order to identify the radical structure of the spin-adducts originating from macromolecules, the usual spin-trapping technique was developed as follows: N2-saturated aqueous solutions of proteins containing sodium formate were X-irradiated (4.5 kGy) in the presence of 2-methyl-2-nitrosopropane (MNP) as a spin-trap. The side-products due to the self trapping of MNP radicals were then removed from the spin-adducts of the proteins by a Sephadex G-25 column. Finally the spin-adducts were enzymatically digested to transform the broad e.s.r. signals due to slow tumbling of nitroxyl radicals to identifiable ones. The e.s.r. spectra obtained for all samples showed that the deaminated radical, R--CH--CO--NH--(R:amino acid side chain), was produced. Furthermore, polyacrylamide gel electrophoresis of the irradiated protamine and histone H1 indicated reduction of molecular size. These results confirm that hydrated electrons react with proteins and induce the deamination reaction which leads to main-chain scission.

Radiolysis of carbohydrates as studied by ESR and spin-trapping. II: glycerol-d8, xylitol, dulcitol, D-sorbitol and D-mannitol

Abstract Studies concerning the radicals produced in glycerol by reactions with OH radicals have been carried out by investigating deuterated glycerol (glycerol-d8) by spin-trapping with 2-methyl-2-nitrosopropane. Free radicals produced in linear carbohydrates such as xylitol, dulcitol, D-sorbitol and D-mannitol by reactions with OH radicals as well as by direct γ-radiolysis have been also investigated by spin-trapping. The ESR spectra of the spin-trapped radicals were analysed on the basis of the results from ESR and spin-trapping experiments on glycerol and deuterated glycerol, and the formation of three radical species, CHO-CH-, CH2-CO- and HO-CH-, due to both OH reactions and direct γ-radiolysis was confirmed for all compounds. The presence of a radical, -CO-CH-, was detected for xylitol, D-sorbitol and D-mannitol. General reactions processes induced by OH reactions or γ-radiolysis in the solid state are discussed.

Radiolysis of carbohydrates as studied by ESR and spin-trapping—I: Glycerol and α-glycerol-1-phosphate

Abstract Free radicals produced in glycerol by reactions with OH radicals and in glycerol phosphate by reactions with OH radicals as well as by direct ψ-radiolysis have been investigated by spin-trapping with t-nitrosobutane (tNB). The presence of two radical species was established for glycerol. Three types of radicals were spin-trapped for glycerol phosphate when OH radical reactions were studied. Two of them were analogous to the radicals obtained for glycerol. On the other hand, when glycerol phosphate was ψ-irradiated in the polycrystalline state and subsequently dissolved in an aqueous solution containing tNB, the spin-trapped radicals are different from those due to reactions between the molecule and OH radicals. The implications of these results are discussed in relation to the indirect and direct mechanisms of the cleavage of carbon-phosphate ester bond in aqueous solution as well as in the solid state.

Electron transfer between protein and DNA in gamma-irradiated deoxyribonucleoprotein

When deoxyribonucleoprotein-proflavine complexes were studied by electron spin-resonance spectroscopy following gamma-irradiation, it was found that stable free radicals were not formed at random on the complex but were preferentially located on proflavine. Since proflavine intercalalated to DNA bases serves as a final acceptor of electrons liberated by ionization, the result of our experiment was regarded as suggesting that the electron transfer from the protein moiety to the DNA moiety occurred in the irradiated deoxyribonucleoprotein.

E.S.R. study on the mechanism of the radiosensitization by nitroxide free radicals

Abstract The reaction of an organic nitroxyl free radical (RNO·) radiation-induced thymine radicals in dilute aqueous solution has been studied at room temperature under O 2 , N 2 or N 2 O. By using E.S.R. and U.V.-spectroscopy it has been found that RNO· react effectively with OH-induced thymine radicals [T(OH)·] under anoxic conditions, while it did not react with T(OH)· under aerobic conditions. The results lead to the conclusion that RNO· does not contribute to cell death in the presence of oxygen.

Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea

1. The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system. 2. The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation. 3. The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range. The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively.

Structural changes of deoxyribonucleoprotein fibres following γ-irradiation under aerobic and hypoxic conditions

The DNP fibres γ-irradiated under aerobic condition showed a reduction of their diameter, while no remarkable changes were observed in the DNP fibres irradiated under hypoxic condition by scanning electron microscopy.