Trace detection of hydroxyl radicals during the redox cycling of low concentrations of diaziquone: A new approach

Quantifying oxygen radicals that arise during the redox cycling of quinone- containing anticancer agents such as diaziquone (AZQ) has been difficult, a s has been their detection at low drug concentrations. This is due to the f act that EPR spin trapping, the method most often used for (OH)-O-. detecti on, requires the use of high drug concentrations. Using a new highly sensit ive technique that employs a fluorescamine-derivatized nitroxide, we show t hai low levels of NADPH-cytochrome P450 reductase (4.25 mu g/ml) catalyze t he production of hydroxyl radicals at very low, clinically relevant AZQ con centrations. Thus, at this enzyme concentration, we were able to detect a r ate of 0.10 nM s(-1) hydroxyl radical production by 5 mu M AZQ, a clinicall y relevant concentration. The Michaelis-Menten constants for AZQ-mediated h ydroxyl radical production are: K-M = 10.7 +/- 1.4 mu M, and V-max = 5.2 +/ - 0.9 X 10(-8) M s(-1) (mg protein)(-1). Experiments employing catalase, su peroxide dismutase, and NADPH-cytochrome P450 reductase, confirm the previo usly deduced conclusions from high drug concentrations, that is, that at lo w concentrations. AZQ acts to shuttle reducing equivalents from the enzyme to oxygen, thus generating the redox cycle. The data presented here suggest that the levels and locations of redox active metal ions may be the princi pal controlling factor in the pathway of AZQ activity that involves oxidati ve stress. (C) 2000 Elsevier Science Inc.