Koch C J, Stobbe C C, Hettiaratchi P
University of Pennsylvania, Philadelphia 19104.
Int J Radiat Oncol Biol Phys. 1989 Apr;16(4):1025-7. doi: 10.1016/0360-3016(89)90908-5.
Radiosensitization of hypoxic V79 Chinese hamster cells by 0.5 mM misonidazole at approximately 0-4 degrees C is substantially enhanced by pretreating the cells overnight with 0.1 mM buthionine sulfoximine, which lowers the cellular glutathione content to 5% of control values (from 4 mM to approximately 0.2 mM). The enhanced sensitization is reversed by concentrations of exogenous cysteine that are much lower (0.02 mM) than the original glutathione content. Reduced Co-enzyme A affords reversal of the enhancing effect at concentrations of about 1 mM. Sodium ascorbate gives no protection at all even at concentrations of 2 mM. The intracellular concentration of the reducing agents was measured using a spin-through oil technique. There was no diffusion of Co-A (MW greater than 750) or ascorbate (excluded by charge) into the cells. In contrast, cysteine was rapidly concentrated by factors of 4-10, even at the low temperatures used. Extracellular ascorbate's inability to radioprotect argues against electron transfer across the cell membrane as a mechanism for radioprotection. This mechanism could have explained the ability of exogenous thiols to radioprotect in former studies using glutathione, and in the present studies using Co-A. The potential of cysteine to be concentrated by cells poses a problem in the interpretation of "exogenous protection" by non-diffusing thiols, since trace contamination by cysteine could lead to the actual protection observed. Cysteine could also be formed by exchange reactions of exogenous thiols with the disulfide of cysteine, present in all media formulations.
在约0 - 4摄氏度下，用0.5 mM米索硝唑对缺氧的V79中国仓鼠细胞进行放射增敏作用，通过用0.1 mM丁硫氨酸亚砜胺对细胞进行过夜预处理可显著增强，该处理可将细胞内谷胱甘肽含量降低至对照值的5%（从4 mM降至约0.2 mM）。增强的增敏作用可被比原始谷胱甘肽含量低得多（0.02 mM）的外源性半胱氨酸浓度逆转。还原型辅酶A在约1 mM的浓度下可逆转增强作用。即使在2 mM的浓度下，抗坏血酸钠也完全没有保护作用。使用自旋穿油技术测量了还原剂的细胞内浓度。辅酶A（分子量大于750）或抗坏血酸（因电荷被排斥）没有扩散到细胞中。相比之下，即使在使用的低温下，半胱氨酸也会迅速浓缩4 - 10倍。细胞外抗坏血酸无法进行辐射防护，这与电子跨细胞膜转移作为辐射防护机制的观点相悖。该机制本可解释在以前使用谷胱甘肽的研究以及在目前使用辅酶A的研究中外源性硫醇进行辐射防护的能力。半胱氨酸被细胞浓缩的可能性给非扩散性硫醇的“外源性保护”解释带来了问题，因为半胱氨酸的微量污染可能导致观察到的实际保护作用。半胱氨酸也可能由外源性硫醇与所有培养基配方中存在的半胱氨酸二硫化物的交换反应形成。
