Intracellular localization of radioactively labeled misonidazole in EMT-6 tumor cells in vitro

Abstract

Misonidazole, as an electron-affinic drug, is capable of accepting electrons from ascorbate, NADPH and a number of enzymes in anoxiic reaction mixtures such as cellular homogenates and microsomal fractions. Although the intracellular location of many of the above electron donors has been established, (usually in microsomes and mitochondria), the sites of covalent binding of metabolically activated misonidazole and its catabolites to cellular macromolecules remain to be elucidated. The biological effects of misonidazole differ quantitatively and possibly qualitatively when cells are incubated in air vis-a-vis hypoxic conditions. Differences in uptake of labeled misonidazole by hypoxic or aerobic cells may render this radiosensitizing agent of value in the clinical management of certain tumors. We are currently attempting to establish the intracellular sites of covalent binding of radioactively labeled misonidazole and its catabolites in EMT-6 tumor cells. Single cell suspensions of EMT-6 tumor cells labeled with 14C-Misonidazole in hypoxic conditions were homogenized to yield nuclear and cytoplasmic fractions, which were assayed for acid-precipitable radioactivity. The distribution of label bound to various intracellular molecular classes, in both the acid soluble and macromoleculer fractions, was determined for EMT-6 cells labeled in air and hypoxia.