The use of 1H spin echo NMR and HPLC to confirm doxorubicin induced depletion of glutathione in the intact HeLa cell

Abstract

Summary The effects of doxorubicin on the cellular biochemistry of the HeLa cell using 1H spin echo nuclear magnetic resonance spectroscopy (NMR) of the intact and viable cell in conjunction with dual wavelength HPLC of cell lysates is reported. Directly dose-related changes were observed in lactate and reduced glutathione concentration. Doxorubicin induces a time-dependent depletion of the cytosolic pool of glutathione and a change in the glycolytic pattern of the cell. The glutathione depletion could be partially reversed by controlled pre-treatment of the cells with N-acetylcysteine and cysteine, the protection being linked to the intracellular concentration of the thiol.

Modelling changes in glutathione homeostasis as a function of quinone redox metabolism

“…For example, lung cancer cell-lines show different sensitivities to doxorubicin in the form of GSH depletion, with A549 and GLC 4 210(S) cells experiencing approximately 50% and 64% GSH depletion after a 12 hour exposure to 70 nM and 5 nM (per million cells) respectively 37 . However, HeLa cells are much more sensitive, with 2.5 nM (per million cells) of doxorubicin resulting in up to 80% GSH depletion for the same exposure time 38 . Some cell-types are much less sensitive to doxorubicin-induced GSH depletion.…”

Menadione-resistant Chinese hamster ovary cells have an increased capacity for glutathione synthesis

“…It is possible to tune out signals arising from large molecules to produce a spectrum composed solely of small molecules, of which GSH is an important example. NMR has proved useful in the non-invasive measurement of thiol redox status (Livesey et al, 1992), as alteration of the GSH-GSSG ratio is easily visualized (McKay et al, 1986;Al-Kabban et al, 1988). Using this approach, it is possible to observe subtle changes in glutathione chemistry.…”