Cytotoxicity of weak electrolytes after the adaptation of cells to low pH: role of the transmembrane pH gradient

Abstract

Summary 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.Keywords: low pH; cell adaptation; pH gradient; weak electrolytes; cytotoxicity As discussed recently (Gerweck and Seetharaman, 1996), the cellular transmembrane pH gradient is significantly decreased, or even reversed, in human solid tumours compared with the corresponding normal tissue. While the intracellular pH (pHi) is similar in both tissues (7.0-7.2 in normal and 7.1-7.3 in tumour tissue), the extracellular pH (pHe) is, on average, 0.2 pH units higher in normal tissues and 0.2-0.6 lower in tumours than pHi. This in vivo difference in cellular pH gradients provides a rationale for the selective intracellular accumulation of various lipophilic drugs, which are weak electrolytes, in tumours or normal tissues.In solution, such chemicals exhibit both a neutral and an ionized form, and the balance of...

Tumor pH controls the in vivo efficacy of weak acid and base chemotherapeutics

“…The results obtained in the present study are qualitatively and quantitatively similar to our previous in vitro studies (13,14). In these in vitro studies, chlorambucil and doxorubicin cytotoxicity and intracellular doxorubicin accumulation were evaluated in cells that were cultured under normal physiologic pH conditions (pH 7.4) as well as in the same cells cultured at low extracellular pH (pH 6.8; i.e., pH condition that mimics the tumor microenvironment).…”

“…It is to be noted that the above analysis is based on the assumption that a linear increase in intracellular drug concentration results in an exponential decrease in the tumor cell surviving fraction, and that surviving tumor cells grow exponentially as shown in Fig. 1A and B. A linear-log relationship between cell drug concentration and cell surviving fraction assessed by colony formation assays has previously been shown by ourselves and others for doxorubicin and chlorambucil over a substantial drug concentration range (13,14,22,23,26).…”

The pH partition theory predicts the accumulation and toxicity of doxorubicin in normal and low-pH-adapted cells

“…As previously described, low-pH-adapted cells are resistant to changes in intracellular pH upon reduction of extracellular pH (Kozin and Gerweck, 1998). Therefore, at a particular extracellular pH, different pH gradients are obtained in the two cell types, whereas at a similar intracellular pH the extracellular pH substantially differs.…”

“…This technique, with some modification, has frequently been used by others and is described in detail elsewhere (Chu and Dewey, 1988;Fellenz and Gerweck, 1988;Kozin and Gerweck, 1998). Briefly, trypsinized cell suspensions (5-10 × 10 5 cells ml -1 ) were concurrently labelled with [ 3 H]water and [ 14 C]DMO or [ 14 C]inulin.…”

Breast Cancer Gene Therapy: Development of Novel Non-Invasive Magnetic Resonance Assay to Optimize Efficacy

“…This though intra and extra cellular concentrations are variable and molecule is restricted to the extracellular compartment only may not always be resolved [121,122]. 19 F NMR agents [107,110], but combination with 3 1 P NMR of Pi to determine pHi has been used to reveal the transmembrane pH can provide a much wider chemical shift range and in some grdienteine mohasbeenuse d tum oreveal [111] [112][113][114][115][116]. However, the pH gradient (ApH) is often small and distinctly variable, and thus, the tumor Several metal ions are thought to play key roles in microenvironment may need to be manipulated to further cellular physiological processes.…”