“…First, let us discuss the additional mechanisms of quinone cytotoxicity. Although dicoumarol possesses several modes of action in the cell ([43], and references therein), our previous and current data show that in MH22a cells under our experimental conditions, it behaved as an NQO1 inhibitor: it decreased the cytotoxicity of aziridinyl-substituted quinones which stems from NQO1-catalyzed reductive activation [37], and increased the cytotoxicity of tetramethyl-1,4-benzoquinone and 9,10-phenanthrene quinone (Figure 6). In this case, NQO1 converts quinones into relatively stable hydroquinones that can be glucuronated or sulphonated and excreted from the cell ([44], and references therein).…”
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.
“…First, let us discuss the additional mechanisms of quinone cytotoxicity. Although dicoumarol possesses several modes of action in the cell ([43], and references therein), our previous and current data show that in MH22a cells under our experimental conditions, it behaved as an NQO1 inhibitor: it decreased the cytotoxicity of aziridinyl-substituted quinones which stems from NQO1-catalyzed reductive activation [37], and increased the cytotoxicity of tetramethyl-1,4-benzoquinone and 9,10-phenanthrene quinone (Figure 6). In this case, NQO1 converts quinones into relatively stable hydroquinones that can be glucuronated or sulphonated and excreted from the cell ([44], and references therein).…”
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.
“…For β-lap-treated astrocytes a strong accumulation of GSSG was observed, indicating that for those conditions the rate of GSH oxidation by GPx is strongly exceeding the rate of GR-mediated GSSG reduction as previously reported for astrocytes that had been exposed to acute or chronic H 2 O 2 -stress [ 23 , 24 ]. One consequence of the strong accumulation of GSSG in β-lap-treated astrocytes is the export of GSSG, which is mediated by Mrp1 [ 30 , 33 ] and has previously been reported for conditions that induce severe oxidative stress in cultured astrocytes [ 25 , 26 , 30 , 33 ]. Fig.…”
Section: Discussionmentioning
confidence: 94%
“…The protein content of the cultures was 148 ± 23 µg/well. Significant differences (ANOVA) compared to the data obtained for the control condition are indicated by asterisks written in the colours of the respective symbols (***p < 0.001) disadvantage that also the export of GSH, GSSG and GSHconjugates from astrocytes via Mrp1 is inhibited [26].…”
Section: Discussionmentioning
confidence: 99%
“…This was achieved by the application of 30 µM dicoumarol which completely prevented NQO1-dependent β-lap-mediated ROS formation and GSSG accumulation. However, application of such high micromolar concentrations of dicoumarol has the disadvantage that also the export of GSH, GSSG and GSH-conjugates from astrocytes via Mrp1 is inhibited [ 26 ].…”
Section: Discussionmentioning
confidence: 99%
“…In addition, GSH can be conjugated to xenobiotics by glutathione-S-transferases to GSH-conjugates [17]. In unstressed astrocytes, hardly any GSSG is detectable, but several compounds have been described to induce GSH oxidation to GSSG which is subsequently exported from the cells, including peroxides [23], catecholamines [24] as well as quinones such as menadione [25,26]. Depletion of cellular GSH in astrocytes was also reported for cells that had been exposed to alkylating substances like iodoacetate [27], 3-bromopyruvate [28] or dialkyl-fumarates [29].…”
β-lapachone (β-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to β-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to β-lap, cultured primary rat astrocytes were incubated with β-lap for up to 4 h. The presence of β-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of β-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM β-lap after a 4 h incubation. Exposure of astrocytes to β-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The β-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to β-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of β-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of β-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.
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