Fig. 1. Nitrofurantoin redox cycling and ROI generation. The one-electron reduction of nitrofurantoin by enzymes such as cytochrome P450 reductase generates the nitrofurantoin radical. In the presence of oxygen, the radical disproportionates to form superoxide anion and the parent compound. Adapted from Minchin et al. [10].

Fig. 2. Effects of nitrofurantoin (NFT) on ROI production by recombinant cytochrome P450 reductase (P450 OR). (A) Nitrofurantoin-induced H₂O₂ production. Enzyme reactions were run in absence or presence of increasing concentrations of nitrofurantoin. Accumulation of H₂O₂ in the assay mixes was quantified using the Amplex red/horseradish peroxidase reaction (n = 3 ± SE). (B and C) Nitrofurantoin-induced hydroxyl radical and superoxide production. Reactions were run in the absence or presence of 25 or 100 μM NFT. Hydroxyl radicals in enzyme assays were quantified by the formation of 2-hydroxyterephthalate (2-OH TPT) derived from terephthalate (n = 2 ± SD). In some assays, catalase (400 U/ml) or DMSO (70 mM) was added to prevent hydroxyl radical formation. Note that the generation of hydroxyl radicals was dependent on the presence of redox-active iron. Superoxide in the enzyme assays was quantified as the formation of 2-hydroxyethidium derived from dihydroethidium. In some of the assays, SOD (350 U/ml) was added to inhibit superoxide formation. C shows HPLC tracings of 2-hydroxyethidium formation. One of two representative experiments is shown. All enzyme reaction mixes contained 0.5 mM NADPH. p < 0.01 compared to rat P450 OR without NFT, # p < 0.05 and ## p < 0.01 compared to rat P450 OR with 100 µM NFT.

Fig. 3. Nitrofurantoin-stimulated NADPH depletion by recombinant cytochrome P450 reductase. Reaction mixes containing 0.1 mM NADPH were analyzed in 1-ml spectrophotometer cuvettes. Absorbance spectra were recorded at 2.5-min intervals. (Top) Nitrofurantoin-stimulated NADPH depletion; 50 μM nitrofurantoin was added directly to the cuvette. (Bottom) NADPH metabolism in the enzyme assay in the absence of nitrofurantoin.

Fig. 4. Effects of inhibitors on nitrofurantoin-induced H₂O₂ production via recombinant cytochrome P450 reductase. H₂O₂ formation was assayed in reaction mixes containing 0.5 mM NADPH, 100 μM nitrofurantoin, and increasing concentration of diphenyleneiodonium (DPI), potassium cyanide (KCN), dicoumarol, or l-NAME. Control rate is 93.4 ± 11.1 nmol H₂O₂/mg protein/min; n = 3 ± SE. p < 0.01 compared to control.

Fig. 5. Effects of nitrofurantoin on ROI production by CHO-WT and CHO-OR cells. (A and B) Nitrofurantoin-induced H₂O₂ production with lysates from CHO-WT cells (A) and CHO-OR cells (B). H₂O₂ production in the assay mixes was quantified in absence or presence of nitrofurantoin every 2.5 min for 30 min (n = 3 ± SE). Constitutive production of hydrogen peroxide was 8 nmol/30 min/mg protein for CHO-WT cells and 21 nmol/30 min/mg protein for CHO-OR cells. (C and D) Generation of hydroxyl radicals and superoxide anion in reaction mixes stimulated by nitrofurantoin. Reactions were run in the absence or presence of 25 or 100 μM nitrofurantoin (NFT). Hydroxyl radicals in enzyme assays were quantified by the formation of 2-hydroxyterephthalate (2-OH TPT) (n = 2 ± SD). In some assays, catalase (400 U/ml) or DMSO (70 mM) was added to prevent hydroxyl radical formation. Superoxide in the enzyme assays was quantified by the formation of 2-hydroxyethidium from dihydroethidium. In some of the assays, SOD (350 U/ml) was added to prevent superoxide formation. D shows HPLC tracings of 2-hydroxyethidium formation. One of two representative experiments is shown. p < 0.05 and p < 0.001 comparing CHO-WT with CHO-OR cells.

Fig. 6. Cytochrome P450 reductase activity, nitrofurantoin (NFT)-induced redox cycling, and cytotoxicity in different cell lines. (Top) Correlation between cellular cytochrome P450 reductase activity and nitrofurantoin-induced H₂O₂ production. Cytochrome P450 reductase activity was measured using a cytochrome c reduction assay. The V_max for nitrofurantoin-induced H₂O₂ generation (Table 1) was used to compare nitrofurantoin-induced redox cycling with cellular cytochrome P450 reductase activity. The inset shows the same data with CHO-OR cells omitted. Note the positive correlation between cytochrome P450 reductase activity and nitrofurantoin-induced redox cycling. (Bottom) Lack of correlation between nitrofurantoin-induced redox cycling and cellular cytotoxicity. Redox cycling capacity of the cell lines was plotted against the concentration of nitrofurantoin inhibiting growth by 50% (IC₅₀) in each cell line (Table 3). (● PC-3, ○ CHO-WT, ▼ HL-60, C2, ■ S-180, □ HT-29, ♦ HeLa, ◊ B16, ▲ RAW 264.7, MLE 15, CHO-OR).

Fig. 7. Intracellular ROI production in CHO-WT and CHO-OR cells. DCFH-DA was used to quantify intracellular ROI generation. Cells were incubated with 5 µM DCFH-DA at 37 °C for 3 h in the absence (solid lines) or presence (dashed lines) of 500 μM nitrofurantoin. Cellular fluorescence was analyzed by flow cytometry. One of three representative experiments is shown. The mean fluorescence of CHO-WT control peak is 1.01 ± 0.04 (n = 3 ± SE) and 7.25 ± 0.28 after nitrofurantoin treatment. For CHO-OR cells, the mean fluorescence of the control peak is 1.13 ± 0.03, 5.01 ± 0.21 for subpeak 1, and 66.40 ± 4.49 for subpeak 2 after nitrofurantoin treatment.

Fig. 8. Growth inhibition by nitrofurantoin. Cells were seeded in six-well plates and allowed to adhere overnight. The medium was then replaced with growth medium supplemented with increasing concentrations of nitrofurantoin. Cell growth was assayed after 4–5 days. (Left) Comparison of the growth inhibitory activity of nitrofurantoin in CHO-WT and CHO-OR cells. (Right) Comparison of the growth inhibitory activity of nitrofurantoin in C2 and S-180 cells.

Kinetic parameters of nitrofurantoin-stimulated H₂O₂ generation with CYP 450 reductase and microsome-containing fractions from different cell types

Effects of nitrofurantoin on the kinetic constants for NADPH with recombinant CYP 450 reductase and microsome-containing fractions from different cell types

Cytochrome c reductase in microsome-containing fractions from different cell types and ability of nitrofurantoin to inhibit cell growth

Kinetic parameters for nitrofurantoin in different fractions of HT 29 and MLE 15 ceslls