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

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.

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Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1)

International Journal of Molecular Sciences ◽

10.3390/ijms21228754 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8754

Author(s):

Aušra Nemeikaitė-Čėnienė ◽

Jonas Šarlauskas ◽

Lina Misevičienė ◽

Audronė Marozienė ◽

Violeta Jonušienė ◽

...

Keyword(s):

Human Colon ◽

Redox Cycling ◽

Geometric Average ◽

Hct 116 ◽

Radical Generation ◽

Electron Reduction ◽

Catalyzed Reactions ◽

Hct 116 Cells ◽

Derivatives Of

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21093234 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3234 ◽

Cited By ~ 2

Author(s):

Mindaugas Lesanavičius ◽

Alessandro Aliverti ◽

Jonas Šarlauskas ◽

Narimantas Čėnas

Keyword(s):

Plasmodium Falciparum ◽

Redox Cycling ◽

Single Electron ◽

Mechanistic Study ◽

Reductive Activation ◽

Midpoint Potential ◽

Quinone Reduction ◽

Rate Limiting Step ◽

Electron Reduction ◽

Intrinsic Reactivity

Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by PfFNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (E17). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of PfFNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar “intrinsic” reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.

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

Chemija ◽

10.6001/chemija.v31i3.4291 ◽

2020 ◽

Vol 31 (3) ◽

Author(s):

Aušra Nemeikaitė-Čėnienė ◽

Jonas Šarlauskas ◽

Violeta Jonušienė ◽

Lina Misevičienė ◽

Audronė Marozienė ◽

...

Keyword(s):

Mammalian Cell ◽

Water Distribution ◽

Reduction Rate ◽

Human Colon ◽

Nitroaromatic Compounds ◽

Single Electron ◽

Cell Cytotoxicity ◽

Electron Reduction ◽

Adrenodoxin Reductase ◽

Dt Diaphorase

Frequently, the aerobic mammalian cell cytotoxicity of nitroaromatic compounds (ArNO2) increases with their single-electron reduction potential (E17), thus reflecting the relationship between their enzymatic single-electron reduction rate and E17. This shows that the main factor of ArNO2 cytotoxicity is redox cycling and oxidative stress. In this work, we found that the reactivity of a series of nitrobenzenes, nitrofurans and nitrothiophenes towards single-electron transferring NADPH:cytochrome P-450 reductase and adrenodoxin reductase/adrenodoxin increases with their E17. However, their cytotoxicity in mouse hepatoma MH22a and human colon carcinoma HCT-116 cells exhibited a poorly expressed dependence on E17. The correlations were significantly improved after the introduction of compound octanol/water distribution coefficient at pH 7.0 (log D) as a second variable. This shows that the lipophilicity of ArNO2 enhances their cytotoxicity. The inhibitors of cytochromes P-450, α-naphthoflavone, isoniazid and miconazole, and an inhibitor of DT-diaphorase, dicoumarol, in most cases decreased the cytotoxicity of several randomly chosen compounds. This shows that the observed cytotoxicity vs E17 relationships in fact reflect the superposition of several cytotoxicity mechanisms.

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Enzymatic single-electron reduction and aerobic cytotoxicity of tirapazamine and its 1-oxide and nor-oxide metabolites

Chemija ◽

10.6001/chemija.v29i4.3843 ◽

2018 ◽

Vol 29 (4) ◽

Cited By ~ 1

Author(s):

Jonas Šarlauskas ◽

Aušra Nemeikaitė-Čėnienė ◽

Audronė Marozienė ◽

Lina Misevičienė ◽

Mindaugas Lesanavičius ◽

...

Keyword(s):

Side Effects ◽

Anticancer Agent ◽

Redox Cycling ◽

Nitroaromatic Compounds ◽

Single Electron ◽

Reductive Activation ◽

Cell Nuclei ◽

Electron Reduction ◽

Phenylene Diamine

Aerobic cytotoxicity of 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ), a bioreductively activated hypoxia-specific anticancer agent, is responsible for TPZ side effects in chemotherapy. In order to clarify its mechanisms, we examined the aerobic cytotoxicity of TPZ and its main metabolites, 3-amino-1,2,4-benzotriazine-1-oxide and 3-amino-1,2,4-benzotriazine in murine hepatoma MH22a cells, and their reduction by NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP+ reductase (FNR). Analogous studies of several quinones and nitroaromatic compounds with similar values of single-electron reduction midpoint potentials (E17) were carried out. In single-electron reduction by P-450R and FNR, the reactivity of TPZ and its monoxide was similar to that of quinones and nitroaromatics, and increased with an increase in their E17. The cytotoxicity of TPZ and its metabolites possessed a prooxidant character, because it was partly prevented by an antioxidant N,N’-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. Importantly, the cytotoxicity of TPZ and, possibly, its 1-N-oxide, was much higher than that of quinones and nitroaromatics with similar values of E17 and redox cycling activities. A possible additional factor in the aerobic cytotoxicity of TPZ is its reductive activation in oxygen-poor cell nuclei, leading to the formation of DNA-damaging species similar to those forming under hypoxia.

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