Kozin S V, Gerweck L E
Edwin L Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA.
Br J Cancer. 1998 May;77(10):1580-5. doi: 10.1038/bjc.1998.260.
Theory suggests that the transmembrane pH gradient may be a major determinant of the distribution of lipophilic weak electrolytes across the cell membrane. The present study evaluates the extent to which this factor contributes to pH-dependent changes in the cytotoxicity of two such chemotherapeutic drugs: chlorambucil and mitoxantrone. Experiments were performed with two cell types of the same origin but exhibiting different pH gradients at the same extracellular pH (pHe): CHO cells cultured under normal physiological conditions (pH 7.4) and acid-adapted cells obtained by culturing under low pH conditions (6.8). Over the pHe range examined (6.0-7.6), the difference between intracellular pH (pHi) and pHe increased with decreasing pHe. Acid-adapted cells were more resistant to acute changes in pHi than normal cells, resulting in substantially larger gradients in these cells. Drug cell survival curves were performed at pHe values of 6.4, 6.8 and 7.4. The cytotoxicity of chlorambucil, a weak acid, increased with decreasing pHe, and low pH-adapted cells were more sensitive than normal cells at the same pHe. In contrast, for the weak base, mitoxantrone, cytotoxicity increased with pHe and was more pronounced in normal cells. As predicted by the theory, the cytotoxicity of both drugs changed exponentially as a function of the pH gradient, regardless of cell type. For mitoxantrone, the rate of such change in cytotoxicity with the gradient was approximately two times greater than for chlorambucil. This difference is probably due to the presence of two equally ionizable crucial groups on mitoxantrone vs one group on chlorambucil. It is concluded that the cellular pH gradient plays a major role in the pH-dependent modulation of cytotoxicity in these weak electrolytes. The data obtained also suggest that a pronounced differential cytotoxicity may be expected in vivo in tumour vs normal tissue. In comparison with normal cells at a pHe of 7.4 (a model of cells in normal tissues), acid-adapted cells at a pHe of 6.8 (a model of cells distal from supplying blood vessels in tumours) were more sensitive to chlorambucil, with a dose-modifying factor of approximately 6, and were more resistant to mitoxantrone by a factor of 14.
理论表明，跨膜pH梯度可能是亲脂性弱电解质在细胞膜两侧分布的主要决定因素。本研究评估了该因素在多大程度上导致了两种此类化疗药物（苯丁酸氮芥和米托蒽醌）细胞毒性的pH依赖性变化。实验使用了两种起源相同但在相同细胞外pH（pHe）下呈现不同pH梯度的细胞类型：在正常生理条件（pH 7.4）下培养的CHO细胞和在低pH条件（6.8）下培养获得的酸适应细胞。在所研究的pHe范围内（6.0 - 7.6），细胞内pH（pHi）与pHe之间的差异随着pHe的降低而增加。酸适应细胞比正常细胞对pHi的急性变化更具抗性，导致这些细胞中的梯度明显更大。在pHe值为6.4、6.8和7.4时进行了药物细胞存活曲线实验。弱酸苯丁酸氮芥的细胞毒性随着pHe的降低而增加，并且在相同pHe下，低pH适应细胞比正常细胞更敏感。相比之下，对于弱碱米托蒽醌，细胞毒性随着pHe的升高而增加，并且在正常细胞中更明显。如理论所预测的，两种药物的细胞毒性均随着pH梯度呈指数变化，与细胞类型无关。对于米托蒽醌，细胞毒性随梯度的变化速率大约是苯丁酸氮芥的两倍。这种差异可能是由于米托蒽醌存在两个同等可电离的关键基团，而苯丁酸氮芥只有一个基团。研究得出结论，细胞pH梯度在这些弱电解质细胞毒性的pH依赖性调节中起主要作用。获得的数据还表明，在体内肿瘤组织与正常组织中可能预期存在明显的细胞毒性差异。与pHe为7.4的正常细胞（正常组织中细胞的模型）相比，pHe为6.8的酸适应细胞（肿瘤中远离供血血管的细胞的模型）对苯丁酸氮芥更敏感，剂量修正因子约为6，对米托蒽醌的抗性则高14倍。
