Mayumi Hori

Effect of Nitric Oxide on γ-Ray-Induced Micronucleus Frequency in RAW264.7 Cells

Abstract Tokuzumi, S., Hori, M., Monobe, M., Hosoi, Y. and Kojima, S. Effect of Nitric Oxide on γ-Ray-Induced Micronucleus Frequency in RAW264.7 Cells. Radiat. Res. 164, 723–732 (2005). The effect of low-dose nitric oxide (NO) on γ-ray-induced micronucleus (MN) frequency was investigated in RAW264.7 cells. Treatment of RAW264.7 cells with 0.25 mM sodium nitroprusside (SNP), a chemical NO donor, reduced the frequency of micronuclei induced by 5 Gy γ rays by 43 to 45% between 3 and 12 h post-treatment. This effect was blocked by carboxy-PTIO, suggesting that NO may play a role in the reduction of radiation-induced MN frequency. To examine possible mechanisms underlying this effect, we first looked at changes in the antioxidant system after SNP treatment. A significant increase in intracellular glutathione (GSH) was seen in SNP-treated cells between 3 and 12 h post-treatment. Depletion of GSH with buthionine sulfoximine (BSO) increased the γ-ray-induced increase in MN frequency. Detailed studies using various inducers of intracellular GSH suggested that GSH induction has a partial role in the reducing effect of NO on the γ-ray-induced MN frequency. Next, the effect of NO on DNA repair and replication systems was examined. Wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK), dose-dependently inhibited the reducing effect of NO, while caffeine, an inhibitor of ATM kinase and ATR kinase, did not. DNA-PK activity was increased by NO treatment. Etoposide, a topoisomerase II inhibitor, dose-dependently blocked the effect of NO in reducing the γ-ray-induced MN frequency. These results suggest that the mechanisms of the effect of NO on the γ-ray-induced MN frequency include elevation of GSH and up-regulation of DNA-PK activity for repairing double-strand breaks. NO may act as a signal for repair systems, e.g. for nonhomologous recombination and for the replication system in S phase, to reduce the MN frequency.

Reduction process of Cr(VI) by Fe(II) and humic acid analyzed using high time resolution XAFS analysis.

The initial reduction behavior of Cr(VI) to Cr(III) has not been clearly understood due to its rapid reduction reaction. In order to study the reduction process of Cr(VI) in detail, we applied quick X-ray absorption fine structure (QXAFS) analysis to observe how Cr(VI) was reduced to Cr(III) by Fe(II) and humic acid (HA) with time. The Cr(VI) concentration was analyzed every 60s, and the plots of ln(Cr(VI)/Cr(VI)0) versus time were used to evaluate the reduction process based on their linearity. Reduction by Fe(II) showed a linear relation, whereas reduction by HA showed a nonlinear relation. With combined Fe(II) and HA, the linearity was unlike those of Fe(II) and HA individually. The reduction rate was not constant. The structure of Fe(II) produced by HA during the Cr(VI) reduction was investigated by using Mössbauer spectroscopy, which showed that Fe(II) produced by HA reduction of Fe(III) had the same structure as the initial Fe(II). HA can reduce Fe(III) back to Fe(II), and reproduced Fe(II) reduces Cr(VI). For Cr(VI) reduction by combined Fe(II) and HA, each reductant contribute differently: Fe(II) directly contributes to the Cr(VI) reaction, whereas HA reduces both Cr(VI) and Fe(III).

Effect of 5,6,7,8-Tetrahydrobiopterin (BH4) on γ-Ray-induced Micronucleus Frequency in RAW264.7 Cells

Abstract The effect of 5,6,7,8-tetrahydrobiopterin (BH4), an endogenous antioxidant, on γ-ray-induced micronucleus frequency was examined in RAW264.7 cells. Exogenously added BH4 reduced the γ-ray-induced micronucleus frequency. Treatment with 1 mM sepiapterin, a biosynthetic precursor of BH4, also significantly reduced the micronucleus frequency induced by 3-10 Oy y-rays, by 20-35(/o at 1 8 h post-treatment. The reducing cffect of SP on γ-ray-induced micronucleus frequency was blocked by N-acetyl-5-hydroxytryptaminc and methotrexate, which are inhibitors of BH4 biosynthesis. These results suggest that intracellular BH4 biosynthesized from sepiapterin is involved in the reducing effect of SP on γ-ray-induced micronucleus frequency. To examine possible mechanisms underlying this action, changes of intracellular BH4 concentration after sepiapterin treatment were examined. Intracellular BH4 concentration peaked at 6 h and was maintained at a high level until 24 h after sepiapterin treatment. However, the γ-ray-induced micronucleus frequency at 6 h post-treatment with sepiapterin showed no change in comparison with that after irradiation alone. These results indicate that the reducing effect of BH4 on γ-ray-induced micronucleus frequency is not necessarily due to antioxidant activity, but may involve other mechanisms.