Miller A C, Blakely W F
Radiation Biochemistry Department, Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-5145.
Free Radic Biol Med. 1992;12(1):53-62. doi: 10.1016/0891-5849(92)90058-o.
Nitrosoureas inactivate cellular glutathione reductase. N,'N'1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosoureas (BCyNU), a nitrosourea reported to selectively inhibit glutathione reductase (GR) activity, was examined to determine if it could be used as a means to inhibit cellular levels of this enzyme in radiobiology studies. Confirmation of drug-induced inhibition of GR activity was demonstrated using a cell-free model system employing purified GR. Cellular studies with Chinese hamster V79A03 showed that BCyNU decreased cellular glutathione content concomitant with an inhibition of specific GR activity. Under relatively nontoxic conditions, cellular exposure to BCyNU (25 microM, 0.25 h) either before or after radiation treatment, increased cellular radiosensitivity with the optimum time for drug addition being immediately following radiation. At a BCyNU dosage which produced less than or equal to 5% cell toxicity, a marked decrease in radioresistance was characterized as a reduction in both Dq (24 +/- 1.5%) and Do (8 +/- 0.5%) concomitant with a 25 +/- 2% decrease in cellular glutathione reductase (GR) activity. At cytotoxic drug dosages (25 microM, 1 h; cell survival 79 +/- 7%), a marked radiosensitization manifested by a 1.25 +/- .07-fold reduction in the Dq was observed concomitant with a 49 +/- 4% decrease in GR activity. Using cells enriched in different stages of the cell cycle, BCyNU caused cell-age dependent cytotoxicity with preferential killing of cells in the radioresistant late-S-phase, a likely explanation for its radiosensitizing capabilities at high drug dosages. Data obtained at nontoxic drug dosages suggest that GR-inactivation may be an important component of cellular response to free-radical induced damage.
亚硝基脲可使细胞谷胱甘肽还原酶失活。N，N′-1，3-双（反式-4-羟基环己基）-N′-亚硝基脲（BCyNU）是一种据报道可选择性抑制谷胱甘肽还原酶（GR）活性的亚硝基脲，本研究对其进行检测，以确定它是否可作为在放射生物学研究中抑制该酶细胞水平的一种手段。使用含有纯化GR的无细胞模型系统证实了药物诱导的GR活性抑制。对中国仓鼠V79A03进行的细胞研究表明，BCyNU可降低细胞谷胱甘肽含量，同时抑制特异性GR活性。在相对无毒的条件下，在放射治疗前或后让细胞暴露于BCyNU（25 μM，0.25小时），可增加细胞放射敏感性，添加药物的最佳时间是在放射后立即添加。在产生小于或等于5%细胞毒性的BCyNU剂量下，放射抗性显著降低，表现为Dq（24±1.5%）和Do（8±0.5%）均降低，同时细胞谷胱甘肽还原酶（GR）活性降低25±2%。在细胞毒性药物剂量（25 μM，1小时；细胞存活率79±7%）下，观察到显著的放射增敏作用，表现为Dq降低1.25±0.07倍，同时GR活性降低49±4%。使用富集于细胞周期不同阶段的细胞，BCyNU引起细胞年龄依赖性细胞毒性，优先杀死抗辐射的S期后期细胞，这可能是其在高药物剂量下具有放射增敏能力的原因。在无毒药物剂量下获得的数据表明，GR失活可能是细胞对自由基诱导损伤反应的一个重要组成部分。
