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

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.

Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme’s ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound’s well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.