Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells

Abstract

[14C]Misonidazole (MISO) becomes bound to macromolecules of mammalian cells upon hypoxic incubation. Intracellular enzyme processes are implicated since the temperature dependence for this process showed an activation energy of 33.5 kcal/mol. The sensitizer bound to both hypoxic and aerobic cells was associated with the macromolecular fraction and the soluble fraction in the proportion, 23 and 77%, respectively. The initial rate of binding of [14C]MISO to the macromolecular (acid-insoluble) fraction of hypoxic EMT-6 mouse tumor and V-79 hamster cells increased proportionally with the square root of extracellular concentration of MISO up to at least 5mM. High concentrations of dimethyl sulfoxide (an effective OH radical scavenger), allopurinol (an effective inhibitor of xanthine oxidase), and diamide (a chemical which can deplete cellular levels of glutathione) had little or no effect on this metabolism-induced binding process. The addition of high concentrations of exogenous cysteamine to hypoxic cell cultures resulted in almost complete inhibition of binding. Extracellular bovine albumin at high concentration in hypoxic cell cultures had little effect on the production of adducts to cell macromolecules and only small amounts of [14C]MISO were found to bind to the extra-cellular bovine albumin. This result suggests that MISO preferentially binds to molecules within the cell in which it is metabolically activated. In experiments where cells labeled under hypoxic conditions with [14C]MISO were subsequently permitted to proliferate in aerobic monolayers, a half-life of the acid-insoluble addition products of approximately 55 hr was measured. A large number of [14C]MISO adducts (approximately 10(9)/cell) can be generated in hypoxic cells without any evidence of cytotoxicity, and they are slowly cleared from cells. These are favorable characteristics as regards the development of this technique as a marker for hypoxic cells in solid tumors.