Reductive metabolism of the hypoxia marker pimonidazole is regulated by oxygen tension independent of the pyridine nucleotide redox state

Abstract

2-Nitroimidazoles, such as pimonidazole, are reduced in cells with low oxygen tension and are, therefore, used as hypoxia markers. However, the effect of the pyridine nucleotide redox state on pimonidazole reduction is not known. Therefore, livers from fed or fasted rats were perfused with oxygen-saturated buffer containing pimonidazole (400 µM) in the presence and absence of an inhibitor of the mitochondrial respiratory chain, potassium cyanide ; these treatments were used to modulate the mitochondrial and cytosolic pyridine nucleotide redox states. Pimonidazole-induced increases in oxygen uptake over basal values were as follows: fed, 15.1Ϯ 2.4 ; fasted, 4.2 Ϯ 0.8; fedϩ KCN, 32.1Ϯ0.9 ; fastedϩKCN, 0.2 Ϯ 0.2 µmol · g Ϫ1 · h Ϫ1 . However, if NADPH was added in excess, microsomal oxygen uptake due to oxidative metabolism of pimonidazole was independent of treatment. These results indicate that pimonidazole-stimulated O 2 uptake, due predominantly to N-oxidation and glucuronidation, is dependent on the NADPH redox state. In contrast, reduced pimonidazole adducts, detected immunochemically, accumulated in pericentral regions in liver. Increasing the NADH redox state by inhibiting the mitochondrial respiratory chain with KCN decreased protein-bound pimonidazole adducts. Concomitantly, the average O 2 tension of the liver was increased at least 30%. However, KCN had no effect on total pimonidazole adducts detected by ELISA, although both cytosolic (lactate/pyruvate) and mitochondrial (3-hydroxybutyrate/acetoacetate) NADH redox states were elevated by at least a factor of eight. These results indicate that, unlike oxidative metabolism, the pyridine nucleotide redox state does not determine the rate of reductive metabolism of pimonidazole. Instead, the cellular oxygen tension regulates this process. Therefore, even in cases where the supply of reducing equivalents is increased (e.g., ethanol metabolism), accumulation of the reduced bound product of pimonidazole is oxygen dependent in liver.

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