Kinetics of Flavoenzyme-Catalyzed Reduction of Tirapazamine Derivatives: Implications for Their Prooxidant Cytotoxicity

Abstract

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit promising antibacterial, antiprotozoal, and tumoricidal activities. Their action is typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the mechanism(s) of aerobic mammalian cell cytotoxicity of ArN→O performing the parallel studies of their reactions with NADPH:cytochrome P-450 reductase (P-450R), adrenodoxin reductase/adrenodoxin (ADR/ADX), and NAD(P)H:quinone oxidoreductase (NQO1); we found that in P-450R and ADR/ADX-catalyzed single-electron reduction, the reactivity of ArN→O (n = 9) increased with their single-electron reduction midpoint potential (E17), and correlated with the reactivity of quinones. NQO1 reduced ArN→O at low rates with concomitant superoxide production. The cytotoxicity of ArN→O in murine hepatoma MH22a and human colon adenocarcinoma HCT-116 cells increased with their E17, being systematically higher than that of quinones. The cytotoxicity of both groups of compounds was prooxidant. Inhibitor of NQO1, dicoumarol, and inhibitors of cytochromes P-450 α-naphthoflavone, isoniazid and miconazole statistically significantly (p < 0.02) decreased the toxicity of ArN→O, and potentiated the cytotoxicity of quinones. One may conclude that in spite of similar enzymatic redox cycling rates, the cytotoxicity of ArN→O is higher than that of quinones. This is partly attributed to ArN→O activation by NQO1 and cytochromes P-450. A possible additional factor in the aerobic cytotoxicity of ArN→O is their reductive activation in oxygen-poor cell compartments, leading to the formation of DNA-damaging species similar to those forming under hypoxia.

QSARs in prooxidant mammalian cell cytotoxicity of nitroaromatic compounds: the roles of compound lipophilicity and cytochrome P-450- and DT-diaphorase-catalyzed reactions

“…The kinetic measurements were carried out spectrophotometrically using a PerkinElmer Lambda 25 spectrophotometer in the 0.1 M K-phosphate buffer (pH 7.0) containing 1 mM EDTA at 25°C. The enzyme activities determined according to the rate of reduction of 50 µM cytochrome c (∆ε 550 = 20 mM -1 cm -1 ) at substrate concentrations indicated below were close to those reported previously [23]: 39 s . In this case, 0.01% Tween 20 and 0.25 mg/mL bovine serum albumin were added as NQO1 activators.…”

“…Murine hepatoma MH22a cells, obtained from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg, Russia), were grown and maintained at 37°C in DMEM medium, supplemented with 10% fetal bovine serum and antibiotics [23]. In the cytotoxicity experiments, 3.0 × 10 4 /ml cells were seeded in 5-mL flasks in the absence or in the presence of compounds, and were grown for 24 h. The cell viability was determined by Trypan blue exclusion.…”

“…In control experiments, the cell viability was 98.5-99.3%. Human colon adenocarcinoma cells HCT-116, obtained from ATCC (Manassas, VA, USA), were grown and maintained at 37°C in 5% CO 2 in the RPMI 1640 DMEM medium, supplemented with 10% fetal bovine serum, 2 mM l-glutamine and antibiotics [23]. In the cytotoxicity experiments, 1.0 × 10 5 /ml cells were seeded in the absence or in the presence of compounds, and were grown for 48 h. Their viability was determined by staining with crystal violet.…”

Reactions of Plasmodium falciparum Ferredoxin:NADP+ Oxidoreductase with Redox Cycling Xenobiotics: A Mechanistic Study

“…In particular, the lower reactivity of ArNO 2 as compared with quinones and ArN→O, which possess similar E 1 7 values (Figure 3), is explained by their k 22 =~10 6 M −1 s −1 [36], which are much lower than those of quinones and ArN→O,~10 8 M −1 s −1 [36,37].…”

Targeting HIF-1&alpha; Function in Cancer through the Chaperone Action of NQO1

“…[162] β-lapachone Futile cycling involving NQO1 results in reduced cellular concentrations of NAD(P)H contributing to cell death [163,164] Mitomycin C Reduction activates this akylating cytotoxic drug [165,166] Tirapazamine and other heteroaromatic N-oxides Slow reaction. Superoxide also produced [167] Benzofuroxans Reduction by NQO1 may play a minor role in cytotoxicity [168] Nitroaromatics Reactivity correlates with electrode potential [169,170] Aminochrome Reaction is important in protection against Parkinson's Disease and other neurological diseases [171,172] Inhibitors Dicoumarol and derivatives thereof High affinity; competes with NAD(P)H; negatively cooperative; often used in experimental studies; derivatives may be anticancer lead compounds; dissociates NQO1-p53 complexes resulting in increased p53 degradation and inhibition of apoptosis [143,151,154,156,173,174] Curcumin May dissociate NQO1-p53 complexes resulting in increased p53 degradation and inhibition of apoptosis. Other studies suggest it may enhance the NQO1-p53 interaction in vivo [175,176] Resveratrol Potent inhibitor of the related protein NQO2; only weakly inhibits NQO1 [177] Warfarin NQO1 is a secondary target for this anticoagulant [174,178] [190,191] Preprints (www.preprints.org) | NOT PEER-REVIEWED Interaction occurs in response to genotoxic stress.…”