Role of the pentose cycle in oxygen radical-mediated toxicity of the thiol-containing radioprotector dithiothreitol in mammalian cells.
We have shown previously that the thiol-containing radioprotector dithiothreitol (DTT) kills V79 cells in a manner that is dependent on both the concentration of DTT and the medium. The results are consistent with the hypothesis that DTT toxicity is caused by the copper-catalyzed oxidation of DTT, forming H2O2, which in turn produces .OH, the ultimate toxic species, via the metal-catalyzed Fenton reaction. Because it is known that the pentose cycle plays a role in the ability of cells to deal with oxidative stress, the hypothesis that the pentose cycle is involved in the response of cells to DTT is tested in this paper. The results show that toxicity of both DTT and H2O2 in V79 cells is greater in cells exposed to the drugs in medium lacking glucose than in cells in medium containing glucose. Addition of glucose to medium or buffer lacking it decreases DTT- and H2O2-induced cell killing. Studies using cells deficient in glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose cycle, show that cells of the mutant cell lines (E16 and E48) are more sensitive to cell killing by both DTT and H2O2 than are the parental CHO K1D cells when exposed to the drugs in medium containing glucose. However, toxicity does not differ significantly among the three cell lines when they are exposed to DTT or H2O2 in phosphate-buffered saline that lacks glucose. Measurements of pentose cycle activity show that the pentose cycle in K1D cells is stimulated by DTT, with the pattern and drug concentration dependence of the stimulation being similar to that for cell killing. However, the pentose cycle is not stimulated by DTT in G6PD-deficient cell lines. The data are consistent with the hypothesis that the pentose cycle is one of the cellular pathways that mediates the oxidative stress imposed by DTT or H2O2.
Held
K D
KD
Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Tuttle
S W
SW
Biaglow
J E
JE
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004229:Dithiothreitol / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000633:toxicity*; D006878:Hydroxides / Q000633:toxicity*; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D011837:Radiation-Protective Agents / Q000633:toxicity*
D006878:Hydroxides; D011837:Radiation-Protective Agents; C031356:hydroxide ion; D006861:Hydrogen Peroxide; D004229:Dithiothreitol