Original Contribution TRACE DETECTION OF HYDROXYL RADICALS DURING THE REDOX CYCLING OF LOW CONCENTRATIONS OF DIAZIQUONE: A NEW APPROACH BEIBEI LI,* NEIL V. BLOUGH,* and PETER L. GUTIERREZ † *Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA; and † The Marlene and Stewart Greenebaum Cancer Center and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA (Received 22 December 1999; Revised 23 May 2000; Accepted 1 June 2000) Abstract—Quantifying oxygen radicals that arise during the redox cycling of quinone-containing anticancer agents such as diaziquone (AZQ) has been difficult, as has been their detection at low drug concentrations. This is due to the fact that EPR spin trapping, the method most often used for • OH detection, requires the use of high drug concentrations. Using a new highly sensitive technique that employs a fluorescamine-derivatized nitroxide, we show that low levels of NADPH-cytochrome P450 reductase (4.25 g/ml) catalyze the production of hydroxyl radicals at very low, clinically relevant AZQ concentrations. Thus, at this enzyme concentration, we were able to detect a rate of 0.10 nM s -1 hydroxyl radical production by 5 M AZQ, a clinically relevant concentration. The Michaelis-Menten constants for AZQ- mediated hydroxyl radical production are: K M = 10.7  1.4 M, and V max = 5.2  0.9  10 -8 Ms -1 (mg protein) -1 . Experiments employing catalase, superoxide dismutase, and NADPH-cytochrome P450 reductase, confirm the previ- ously deduced conclusions from high drug concentrations, that is, that at low 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 principal controlling factor in the pathway of AZQ activity that involves oxidative stress. © 2000 Elsevier Science Inc. Keywords—Hydroxyl radical, Diaziquone, Nitroxide, High performance liquid chromatography, Fluorescence detec- tion, Fluorescamine, Dimethyl sulfoxide, Electron paramagnetic resonance, Free radicals INTRODUCTION The anticancer agent diaziquone (2,5-diaziridinyl-3,6- bis(carboethoxyamino)-1,4-benzoquinone; AZQ) was originally designed to cross the blood-brain barrier and thus potentially be active against malignancies of the central nervous system [1–5]. The carboethoxyamino groups facilitate transport across the blood-brain barrier, while the aziridines provide bifunctional alkylation to DNA. The quinone moiety places AZQ in a class of agents that undergo bioreductive activation [6 –13]. Thus two modes of action have been postulated for AZQ: (i) alkylation of DNA [14 –16], which is increased upon the bioreduction of the drug [17], and (ii) the production of reactive oxygen species (ROS) during redox cycling [e.g., 6]. Whole cells and purified enzymes can reduce AZQ to the semiquinone (1e - ) or the hydroquinone (2e - ), thus increasing alkylation. Oxidation of the re- duced species leads to formation of the superoxide rad- ical anion (O 2 •- ), hydrogen peroxide, and, in the pres- ence of some trace metals, the highly oxidizing hydroxyl radical ( • OH) [e.g., 6,8,18 –20]. If redox cycle-generated ROS are not quenched, they become implicated in all oxidative stress-related events that take place in the cell. These include, for instance, signal transduction, gene activation, and hydroxyl radical-mediated DNA damage. This latter damage can be of two kinds, one is strand breaks and the other, the mutagenic lesion 8-oxodG. The redox cycling of AZQ has been shown to induce strand breaks [20] and 8-oxodG [21]. There is recent evidence that DT-Diaphorase-mediated reduction of aziridine qui- nones, including AZQ, induce the tumor suppressor gene Address correspondence to: Peter L. Gutierrez, Ph.D., University of Maryland Cancer Center, 655 West Baltimore Street, Baltimore 21201 MD, USA; Tel: (410) 328-3685; Fax: (410) 328-6559; E-Mail: pgutierr@som.umaryland.edu. Free Radical Biology & Medicine, Vol. 29, No. 6, pp. 548 –556, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter PII S0891-5849(00)00402-0 548