Hirotoshi Maki

Cell Inactivation and DNA Single- and Double-strand Breaks in Cultured Mammalian Cells Irradiated by a Thermal Neutron Beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.

Asymmetrical radical formation in D- and L-alanines irradiated with tritium-β-rays

Radical formation in D- and L-alanines was studied using ESR after internal3H-β-irradiation under the situation in which the contribution of Bremsstrahlung to form the radicals is assumed to be considerably less than the case of9 0Y-β-irradiation. It was demonstrated that the relative radical concentration by the β-rays was distinguishably higher in D-alanine than in L-alanine. Thus the asymmetry found in these experiments in the radical formation of the alanines may be attributed to the different interaction between the polarized electrons and the two enantiomers.

Synthesis of195mPt radiolabelled cis-diamminedichloroplatinum/II/ of high chemical and radiochemical purity using high performance liquid chromatography

An improved method is described for the synthesis of195mPt-radiolabelled cis-diamminedichloroplatinum/II. An amount of 10 mg of 95% enriched194Pt was irradiated for 75 h in the hydraulic conveyer of the KUR at a thermal neutron flux of approximately 8.15×1013 n.cm−2.sec−1 and the195mPt-radiolabelled CDDP was purified using HPLC. The chemical yield is 61% its chemical purity is greater than 99.74% the radiochemical purity is nearly 100%, and the specific activity is 7.4×106 Bq mg−1 CDDP/200 μCi mg−1 CDDP/.

Determination of the target volume of HeLa cells treated with platinum-195m radiolabeled trans-diaminedichloroplatinum(II): a comparison with cis-diaminedichloroplatinum(II)

HeLa S-3 cells were treated with 195mPt-radiolabeled trans-diaminedichloroplatinum(II) (TDDP) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with TDDP at 37 degrees C for 1, 2 and 3 h were 163.7, 65.8 and 24.9 microM, respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules was determined after the cells were fractionated using the method of Schneider. In this way, the D0 values given as the drug concentration were substituted for the number of Pt atoms combined with each fraction, then the target volumes, expressed as the reciprocals of the D0 values, were calculated for each fraction. The results suggested that DNA and high molecular weight RNAs (except t-RNA), under some limited condition, could be the target molecules for cell killing by TDDP. The target volumes for DNA were 1.31 x 10(3), 3.01 x 10(3) and 6.26 x 10(3) nucleotides for 1, 2 and 3 h treated cells, respectively. Cell killing effects of TDDP were lower than CDDP by a factor of 39.5, 19.0 and 16.5 for 1, 2 and 3 h treatments at 37 degrees C, respectively, when viewed from the stand point of the target volume, while those from the mean lethal dose (D0) were 17.6, 9.8 and 6.7, respectively.

An approach to the mechanism of the asymmetrical radical formation in yttrium-90-beta-irradiated D- and L-alanines.

Several attempts were made to investigate the mechanism of the asymmetrical radical formation in yttrium-90-beta-irradiated D- and L-alanines which was reported in the preceding paper (1). The experiments demonstrated that the magnitude of the asymmetry was dependent on beta-ray dose, namely the lower the dose the larger the observed difference was, and that no difference could be detected when the alanines were irradiated in aqueous state. The results in the present study seem to that different interaction between the crystal structure of the two enantiomers and polarized beta-rays may be responsible for the observed phenomenon.

The Contribution of the Nuclear Reaction 1H(n, γ)2D to the Yield of DNA Single Strand Breaks in Cultured Mammalian Cells Irradiated by Thermal Neutrons

The yield of single strand breaks (ssb) in DNA of the HeLa S-3 cells after thermal neutron irradiation was examined using the alkaline sucrose gradient method. The contribution of the 1H(n, gamma)2D reaction to the yield of ssb was determined by substituting D2O for H2O in the irradiated medium. Calculation shows that when cells are irradiated in the H2O medium, the per cent contribution of the contaminating gamma-rays, the nuclear reaction 1H(n, gamma)2D and the other nuclear reactions is 31, 44 and 25 per cent respectively assuming additivity of effects. The estimated number of ssb induced by the nuclear reaction 1H(n, gamma)2D was at least 4.4 times greater than that by 60Co gamma-rays at the same absorbed dose. Two possible interpretations are discussed to explain the high efficiency of the 1H(n, gamma)2D reaction for ssb induction.

Selective decomposition of either enantiomer or aspartic acid irradiated with60Co-γ-rays in the mixed aqueous solution with D- or L-alanine

Aqueous solutions of various amino acids were irradiated with60Co-γ-rays, and subsequently the remaining amino acids were analyzed using HPLC. The D37 for the 1 mM glycine and alanine solutions were 1.95×104 and 1.48×104 Gy, respectively. However, when the mixed solutions of glycine and alanine (each in 0.5 mM) were irradiated under the identical condition, the D37 for the glycine decomposition increased to 3.56×104 Gy, while that for alanine decreased to 0.65×104 Gy. A similar phenomenon was observed also in the case of the mixed solutions of aspartic acid and alanine. Namely, aspartic acid was protected from the attack of radiation by the presence of alanine in the solutions. The most interesting finding in this combination experiment is that, when D,L-aspartic acid was irradiated in the presence of L-alanine, the radiation-sensitivity of L-aspartic acid decreased selectively and vice versa. Namely, the asymmetric field induced in the solutions by adding D- or L-alanine might affect the radiodecomposition rate of either aspartic acid. Addition of glycine to D,L-aspartic acid did not bring about the asymmetric decomposition. It seems that some interaction between these amino acid molecules resulted in this effect.

Synthesis of195mPt radiolabeled (−)-(R)-2-aminomethyl-pyrrolidine (1,1-cyclobutanedicarboxylato)-2-platinum(II) monohydrate using high performance liquid chromatography

An experimental method is described for the synthesis of195mPt-radiolabeled (−)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutanedicarboxylato)-2-platinum (II) (DWA2114R). An amount of 10 mg of 95% enriched194Pt was irradiated for 75 h in the hydraulic conveyer of the KUR at a thermal neutron flux of approximately 8.1×1013 n.cm−2.sec−1 and the195mPt-radiolabeled DWA2114R was purified using HPLC. The chemical yield is higher than 65%, its chemical purity is greater than 99.2%, the radiochemical purity is nearly 100%, and the specific activity is 6.7 MBq. mg−1 DWA2114R.

Determination of the target volume of hela cells treated with tetra‐ and hexa‐ chloroplatinates

HeLa S‐3 cells were treated with l95mPt‐radiolabeled tetra‐ and hexa‐chloroplatinums (Pt(II) and Pt(IV)) under various conditions, and the relationship between lethal effect and the number of Pt‐atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with Pt(II) at 37°C for 1, 2 and 3 h were 163.5, 126.2 and 67.3 μM, while those for Pt(IV) were 78.6, 53.4 and 35.9, respectively. By using identically treated cells, the number of Pt‐atoms combined with DNA, RNA and protein molecules were determined after the cell were fractionated using the method of Schneider [ref. 1]. In this way, the Do values given as the drug concentration were substituted for the number of Pt‐atoms combined with each fraction, then, the target volumes expressed as the reciprocals of Do values were calculated for each fraction. The results suggested that DNA and high molecular weight RNAs (except t‐RNA), under some limited condition, can be the target molecules for cell killing by both ...

Lethal Effect and Potentially Lethal Damage Recovery in Cultured Mammalian Cells Irradiated by Neutron-capture Beams

Cell killing and potentially lethal damage (PLD) recovery in Hela cells were examined after irradiation in suspension using a 10B neutron-capture beam. Thermal-neutron irradiation in the medium containing from 0.09 to 9 mM boron-10 (0.9-90 micrograms/g 10B, 90 per cent enriched boric acid) resulted in steeper survival curves than in the boron-free medium. The relative biological effectiveness (r.b.e.), expressed as the Do ratio, increased from 2.2 +/- 0.1 for the control without boron-10 to 5.4 +/- 0.3 in the medium containing 0.9 mM boron-10. The r.b.e. value was 5.2 +/- 0.5 at a boron-10 concentration of 9 mM, and did not increase when the boron-10 concentration was increased from 0.9 to 9 mM. It was suggested that the observed plateau of r.b.e. at boron-10 concentrations higher than 0.9 mM was due to the dominancy of the 10B(n, alpha)7Li reaction for dose accumulation. PLD recovery after the neutron irradiation containing 0.9 mM boron-10 was not observed, while a small effect was observed after irradiation in the thermal neutron beam, although much less than after gamma rays. The results show that the neutron-capture beam is an excellent beam for radiotherapy if the boron-10 concentration in tumour cells could be elevated to about 1 mM.