Archives of Biochemistry and BiophysicsVol. 397, No. 1, January 1, pp. 113–118, 2002doi:10.1006/abbi.2001.2670, available online at http://www.idealibrary.com on

Revisiting the Interaction of the Radical Anion Metaboliteof Nitrofurantoin with Glutathione

Catherine Miller,*,1 Lisa K. Folkes,† Carolyn Mottley,‡ Peter Wardman,† and Ronald P. Mason§*Department of Chemistry, John Carroll University, Cleveland, Ohio 44118; †Gray Laboratory Cancer Research Trust,Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom; ‡Department of Chemistry, Luther College,Decorah, Iowa 52101-1045; and §Laboratory of Pharmacology and Chemistry, National Institute of EnvironmentalHealth Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received September 17, 2001, and in revised form October 22, 2001; published online December 4, 2001

There have been several conflicting reports as to thescavenging nature of glutathione toward the nitroradical anion of the drug nitrofurantoin. We producedthe radical anion enzymatically using the xanthineoxidase/hypoxanthine system at pH 7.4 and pH 9.0 inthe presence of various levels of glutathione from 10 to100 mM and monitored any changes in the radicalconcentration via electron spin resonance spectros-copy. Independent of glutathione concentration, therewas no decrease in the steady-state concentration ofthe radical. In fact, there was an average 30% increasein the concentration of the radical anion, which sug-gests enhanced enzyme activity in the presence of glu-tathione (GSH). These results, together with observa-tions of the effects of glutathione on the stability of theradical anion generated by radiolysis or dithionite,rule out any detectable reaction between the nitro-furantoin radical anion and GSH under physiologi-cally relevant conditions. © 2001 Elsevier Science

Key Words: free radical; glutathione; nitrofurantoin;electron spin resonance; pulse radiolysis; stoppedflow.

Nitrofurantoin (N-(5-nitro-2-furfuryldine)-1-amino-hydantoin) (NFT)2 is a nitroaromatic compound that isused as a urinary antimicrobial agent (1, 2). It belongsto the class of nitroheterocyclic compounds, which in-cludes nitrofurans and nitroimidazoles. These com-

1 To whom correspondence and reprint requests should be ad-dressed at Department of Chemistry, John Carroll University, 20700North Park Boulevard, Cleveland, OH 44118. Fax: (216) 39-1791.E-mail: cmiller@jcu.edu.

2

Abbreviations used: NFT, N-(5-nitro-2-furfuryldine)-1-aminohy-dantoin; ESR, electron spin resonance; GSH, glutathione.

0003-9861/01 $35.00© 2001 Elsevier ScienceAll rights reserved.

pounds been investigated for use in cancer therapy asradiosensitizers and hypoxia-specific cytotoxic agents(3–7). However, these compounds have been associatedwith lung and liver damage (2). The mechanism(s) ofcytotoxicity is not well understood, except that thenitro radical anion is an obligate intermediate, andchemical damage may be linked to its further reductionproducts such as the corresponding nitroso and hydrox-ylamine derivatives (8).

The one-electron reduction of NFT to the nitro radi-cal anion is catalyzed by several intracellular flavopro-tein reductases such as cytochrome P450 reductase,xanthine oxidase, and aldehyde oxidase. The NFT rad-ical anion may also be generated using biochemicalreducing agents such as ascorbate and catecholamines(3, 5, 8).

ArNO2O¡

1e–

enzyme

ArNO2•2 [1]

Once formed, the nitro anion radical disproportionatesunder hypoxic conditions to form the nitroso, hydroni-troxide, and amine derivatives. Under aerobic conditions,in the presence of liver and lung microsomes, NFT un-dergoes redox cycling where the radical anion reducesoxygen to the superoxide radical anion, O2

•2 (9–11).

ArNO2•2 1 O23 ArNO2 1 O2

•2 [2]

This redox cycling, also referred to as “futile metabo-lism” (12), is in competition with an acid-catalyzed,second-order decay pathway of disproportionation (9).

2ArNO2•2 1 2H13 ArNO2 1 ArNO 1 H2O [3]

113

114 MILLER ET AL.

The redox properties of nitroaromatic compoundshave been studied extensively using photolysis (13),pulse radiolysis (14, 15), and electrochemistry, specif-ically, cyclic voltammetry and polarography(16–18).The reduction potentials of nitro compounds are linkedto their biological activities. The reduction potentialdetermines both the ease of electron donation from theenzyme (Reaction [1]) and the rate of Reaction [2] (9,19, 20). Free radicals involved in prototropic equilibriaof coupled electron/proton-transfer reactions [3] haveshown dramatic effects on the chemical reactivity ofthe radical, its stability in aqueous solution, the ratesof electron-transfer, and pH-dependent redox proper-ties (21).

Electrochemical studies of the reduction of nitroaro-matics have used aqueous solutions as well as mixedmedia with added organic solvent (dimethylform-amide) to aid in the dissolution of the drugs. In-depthstudies in aqueous media showed a complex reductionmechanism over a large pH range due to adsorptionand protonations, factors that can be difficult to controlin an electrochemical experiment (18, 22). Thus, mostelectrochemical studies are carried out in a mixed me-dia, often in aprotic solvents, where results still show astrong pH dependence as well as a dependence on theaqueous/organic composition (22).

This pH dependence is in agreement with the elec-tron spin resonance (ESR) studies (10) and pulse radi-olysis studies (23) that examined the nitro radical an-ion of several nitroaromatic compounds at several dif-ferent pH values. Other ESR studies looked at theinteraction of the nitro radical anion with relevantbiological reducing macromolecules, specifically, gluta-thione (GSH) (11). They found no interaction withGSH. However, in a series of mixed media and aproticelectrochemical investigations of several nitro-aro-matic compounds, Squella et al. (24–26) interpretedtheir electrochemical data to conclude that GSH re-acted with the radical anion of a variety of nitro com-pounds. This paper reexamines the reported interac-tion of GSH with nitrofurantoin at pH 7.4 and 9.0 andconcludes that GSH does not chemically react with thenitrofurantoin radical anion.

MATERIALS AND METHODS

Xanthine oxidase (purified from buttermilk, 20.8 Units/mL), hypo-xanthine, NFT, and GSH were obtained from Sigma Chemical Co.(St. Louis, MO). Buffers were prepared using sodium phosphatedibasic heptahydrate, sodium phosphate monobasic, and sodium hy-droxide from Mallinckrodt (Paris, KY).

The NFT radical was generated at room temperature (23°C) usingan enzymatic system of xanthine oxidase and hypoxanthine in 100mM phosphate buffer at pH 7.4 and 100 mM borate buffer at pH 9.0.A stock solution containing 1 mM NFT and 1 mM hypoxanthine in anappropriate buffer was prepared and protected from daylight. A

fresh 200 mM GSH solution was prepared using deoxygenated (bub-bled with N2) buffer. The GSH solution was adjusted to the desired

pH using concentrated NaOH and stored in an airtight container. A2.5-mL aliquot of the drug/hypoxanthine stock was transferred to avial, capped with a septum, and bubbled with N2 for 5 min. Aliquotsof GSH were added to the drug solution to obtain varying GSHconcentrations from 10 to 100 mM. The final volume of samplesolution was adjusted to 5 mL using deoxygenated buffer, givingfinal concentrations of 0.5 mM NFT and 0.5 mM hypoxanthine.Xanthine oxidase was added to initiate the reaction to a final con-centration of 0.1 Unit/mL. The solution was vortexed for 15 s anddrawn by vacuum suction through stainless-steel tubing into aquartz aqueous flat cell positioned in a super high Q cavity of aBruker EMX spectrometer. Spectra were collected at approximately2 and 8 min after the addition of the enzyme to determine thesteady-state concentration of the radical anion. The NFT radicalanion was generated at pH 9.0 using the same procedure.

The steady-state concentration of radical ion was determined us-ing a standard calibration curve prepared from a stable nitroxidesolution of TEMPOL (27). Integrated intensities of the spectra wereobtained using Bruker WIN-EPR software (28). The power and mod-ulation amplitude were appropriately chosen for the radical calibra-tion curve.

The nitro radical anion was also generated by pulse radiolysis (0.5ms, ;59 Gy, ;6 MeV electrons), producing ;40 mM radicals. Solu-tions contained sodium formate (0.1 M), NFT (0.5 mM), GSH (0 or 0.8mM), and N2O-saturated NaOH aqueous solution at pH 8.9; CO2

•2 isthe reducing agent. The equipment has been described (29). Forstopped-flow spectrophotometric experiments, the nitro radical an-ion was generated by mixing sodium dithionite (;30–50 mM, con-centrations after mixing) with ;0.3–0.5 mM NFT in borate buffer,pH 9.1, and GSH was added (0.5 mM) after 2.5 s using a Hi-TechSF-61 DX2 sequential-mix (four-syringe) instrument.

RESULTS AND DISCUSSION

The nitroheterocyclic nitrofurantoin was reduced toits respective nitro anion radical in an anaerobic incu-bation with xanthine oxidase and hypoxanthine. Anincubation of the complete system, 0.5 mM NFT, 0.5mM hypoxanthine, and 0.1 U/mL xanthine oxidase atpH 7.4, shows a multiple-line ESR spectrum indicativeof the nitro radical anion, the parent compound plus anelectron, as shown in Fig. 1A. Control experimentsshowed that the radical production is dependent on thepresence of enzyme (Fig. 1B) and substrate (data notshown). The production of the nitro radical anion wasalso dependent on the xanthine oxidase cofactor hypo-xanthine (Fig. 1C) because it provides a source of re-ducing equivalents. Our interest was in the interactionof the nitro radical anion with GSH. As expected, GSH(100 mM) could not replace hypoxanthine in support-ing nitro radical anion formation (Fig. 1E). At theconcentrations specified, the radical anion signal wasdetected for 13 min at a steady-state concentration.The nitro radical anion presumably decays by dispro-portionation, under hypoxic conditions (8). Althoughthe nitro radical anion also reacts with oxygen, the flatcell is a closed system and all solutions were bubbledwith nitrogen.

The spectra of the NFT radical anion generated atpH 7.4 and pH 9.0 are shown in Figs. 2A and 3A,respectively. Note that the signal is pH dependent, and

the spectra have a higher resolution at pH 9.0. The

115NITROFURANTOIN RADICAL ANION METABOLITE INTERACTION WITH GLUTATHIONE

ESR spectra were simulated at both pH values, allow-ing for the accurate determination of hyperfine split-tings of the radical anion (Table I). At the higher pHvalue, the hyperfine splittings agree well with the lit-erature parameters although these values were deter-mined from the radical anion that was generated usingascorbate in 0.1 M sodium hydroxide (10). This pHdependence of the steady-state ESR spectrum is con-sistent with the fact that xanthine oxidase activityincreases with pH (8) and, as shown in Eq. [3], thedisproportionation reaction is acid catalyzed. At bothvalues, pH 7.4 and pH 9.0, the predominant species isArNO2

•22 because the pKa of the aminohydantoin groupis 7.2 (30). Note that the pKa of the hydantoin N–Hionization in the radical anion will not be identical tothat in the ground state, but very close. Wardman et al.found that a nitroimidazole with a three-carbon satu-rated side chain terminating in N-piperidino had ameasurably different (0.5 shift) N-protonation pK in

FIG. 1. EPR spectra of the nitrofurantoin anion radical producedenzymatically in a 100 mM phosphate buffer, pH 7.4, using 0.5 mMnitrofurantoin, 0.5 mM hypoxanthine, and 0.1 U/mL xanthine oxi-dase under a nitrogen atmosphere. Instrumental conditions were 5.0mW microwave power; 0.20 G modulation amplitude; 655 ms timeconstant; 335 s scan rate; and 5 3 105 gain. (A) Complete system; (B)same as in A, except without xanthine oxidase; (C) same as in A,except without hypoxanthine; (D) same as in C, except with theaddition of 100 mM GSH.

a

the radical compared to the ground state (15).

Fig. 2C shows the effect of the addition of 100 mMGSH at pH 7.4. The pH of the fresh GSH solution wasadjusted to pH 7.4 prior to addition to the mixtures. IfGSH reacted with the nitro radical anion, the ampli-tude of the steady-state signal would be expected todecrease. However, there was no decrease in the am-plitude of the ESR signal and, hence, no decrease in theradical anion concentration. The same trend was foundat pH 9.0. Figure 3C is the spectrum generated uponthe addition of 10 mM GSH at pH 9.0 to the originalincubation mixture. Regardless of whether 10 or 100mM GSH was added to the enzyme/drug system, therewas actually a consistent increase in the amplitude ofthe spectra. The steady-state concentration of the nitroradical anion was measured in the presence of varyingconcentrations of GSH from 10 to 100 mM (Fig. 4).Addition of GSH increased the radical anion concen-tration by 29% (pH 7.0) and 31% (pH 9.0). The fact thatthere was an increase in the steady-state concentrationof NFT•2 suggests that under these conditions GSHwas increasing the enzyme activity. Increasing the

FIG. 2. Effect of GSH on the nitrofurantoin anion radical producedenzymatically in a 100 mM phosphate buffer, pH 7.4, using 0.5 mMnitrofurantoin, 0.5 mM hypoxanthine, and 0.1 U/mL xanthine oxi-dase under a nitrogen atmosphere. (A) Complete system (withoutGSH); (B) simulation of (A) (parameters in Table I); (C) same as in A,except with 100 mM GSH with the pH readjusted to 7.4. Instrumen-

tal conditions were 5.0 mW microwave power; 0.20 G modulationamplitude; 655 ms time constant; and 335 s scan rate.

116 MILLER ET AL.

GSH concentrations beyond 10 mM had no furthereffect on the concentration of NFT•2.

The unreactivity of the enzymatically generatednitrofurantoin anion radical with GSH as measuredby ESR (Figs. 1–3) is entirely consistent with otherexperiments involving nitro anion radical generationeither by radiolytic or chemical reduction. Genera-tion by pulse radiolysis (one-electron reduction by

FIG. 3. Interaction of nitrofurantoin anion radical with GSH at pH9.0. ESR spectra of the nitrofurantoin anion radical produced enzy-matically in a 100 mM phosphate buffer, pH 9.0, using 0.5 mMnitrofurantoin, 0.5 mM hypoxanthine, and 0.1 U/mL xanthine oxi-dase under a nitrogen atmosphere. (A) Complete system (withoutGSH); (B) simulation of (A) (parameters in Table I); (C) same as in(A), except with the addition of 10 mM GSH. The pH of all solutionswas adjusted to pH 9.0. Instrumental conditions were 5.0 mW mi-crowave power; 0.20 G modulation amplitude; 655 ms time constant;and 335 s scan rate.

TABLE I

Hyperfine Coupling Constants for Nitro Radical AnionRadical from Nitrofurantoin (in Gauss)

Assignment pH 7.4 pH 9.0 Ref. (10)

1N (NO2) 10.87 11.03 11.031H (4) 5.77 5.89 5.911H (3) 1.51 1.56 1.531H (HC 5 N) 0.81 1.05 1.001N (HC 5 N) 2.29 2.27 2.302H (.N2 CH22 0.70 0.75 0.76

1N (2N,) 0.170 0.30 0.31

CO2•2) produced the absorbance of NFT•22 with max-

ima at wavelengths .500 nm (where the groundstate does not absorb) occurring at ;605 and 660 nm(e ; 350 M21 cm21). Figure 5 shows that the decay ofthe absorbance over ;1.8 s was, if anything, slightlyslower in the presence of GSH. Sodium dithionitereduces NFT via the SO2

•2 radical anion (31), and anoptical spectrum of NFT•22 identical to that observedusing pulse radiolysis was observed ;1.5 s aftermixing dithionite and NFT. As shown also in Fig. 5,the rate of decay over ;25 s was not greater in thepresence of GSH; the rapid but partial decay occur-ring in the initial phase may be due to reaction withresidual oxygen.

The rate of disproportionation of the radical anionmust be compared to the reaction rate of GSH with theradical anion.

FIG. 4. The steady-state radical anion concentration as a functionof GSH concentration at pH 7.4 and pH 9.0. The radical anion fromnitrofurantoin was generated via xanthine oxidase/hypoxanthine.The pH of the GSH stock was adjusted to the appropriate pH andbubbled with N2 before addition to the NFT/XO solution.

FIG. 5. Decay of absorbance of the nitrofurantoin radical anionafter production by pulse radiolysis (A) or dithionite reduction (B)

and the effect of added GSH (0.8 or 0.5 mM concentrations aftermixing) pH 8.9 or 9.1, respectively.

117NITROFURANTOIN RADICAL ANION METABOLITE INTERACTION WITH GLUTATHIONE

k [GSH] [NFT•2] ,, 2kd [NFT•2 ]2 [4]

k ,, 2kd [NFT•2]/[GSH] [5]

Pulse radiolysis measurements of the nitrofurantoinradical anion at pH 9.0 yielded a disproportionationconstant (k d) of 2k ; 4 3 10 3 M21 s21 (data notshown). This rate constant is of the same order asthat reported by Guissani et al. (32). For example,using the data in Fig. 4, in the presence of 100 mM[GSH] and a radical concentration of 16 mM, anyreaction between these two species would have a rateconstant (k) of 0.64 M21 s21 as an upper limit. Forcomparison, the reactivity of NFT•2 toward oxygenhas a rate constant of approximately 1 3 106 M21 s21.Even under hypoxic conditions (1 mM O2), in thepresence of cellular levels of GSH (2 mM), the reac-tion of NFT•2 and O2 would be at least 200 timesfaster than the reaction with GSH. In normal tissueswith an O2 concentration of 40 mM, oxygen wouldoutcompete GSH by almost a factor of 10,000. Inneither case is the scavenging of the nitrofurantoinradical by GSH kinetically feasible.

There is, thus, no evidence at all, using three in-dependent methods of generation of the radical anionand two methods of detection, that GSH reacts withthe nitrofurantoin radical. The increase in steady-state concentration on adding GSH in the ESR ex-periments may reflect an increase in enzyme activityand/or, more speculatively, a slightly increased life-time arising from GSH removing the radical dispro-portionation product, the nitrosofuran, as soon as itis formed. Reduction of nitroarenes proceeds rapidlythrough to the hydroxylamine via the nitrosoarene,and the latter can very probably be reduced by thenitro anion radical. This reaction may occur concur-rently with disproportionation in some circum-stances (15). Also, it has been shown that nitro-soarenes react rapidly with GSH (33).

The possible contribution to electrochemical mea-surements of rapid reaction between GSH and theproduct of disproportionation of nitro anion radicals isunclear. Interpretation of electrochemical data to de-rive radical lifetimes is clearly less reliable than directobservation of steady-state concentrations or the ab-sorbance of the radical. The data in Fig. 5, consistentwith the ESR observations, prove that the time scale ofany interaction between the nitro anion radical andGSH at levels of the latter found intracellularly (a fewmillimolar) must be, at the very least, on the time scaleof several seconds. In aerated solutions, typical nitro-furan anion radicals disappear in a few tens of milli-seconds (e.g., see data for nifuroxime (9)). Interactionbetween the nitro anion radical and GSH is unlikely to

be of physiological importance.

Depletion of GSH (34, 35) is associated with oxida-tive stress. Nitro-drug toxicity may result from super-oxide formation via radical anion production. However,GSH depletion is caused by reaction with the nitrosoand hydroxylamine species, not with the radical anion(36). These reduced species also react with proteinthiols. Hypoxic toxicity of nitrofurantoin is associatedwith protein alkylation: [14C]nitrofurantoin binds irre-versibly to protein, accompanied by protein thiol deple-tion without formation of GSH–protein mixed disul-fides (37). Thus, GSH depletion has been reported bothin vitro and in vivo (38–41), but our studies show thatthis depletion is not caused by reaction of GSH with thenitro radical anion.

ACKNOWLEDGMENTS

The Clare Boothe Luce Foundation, and The Cancer ResearchCampaign supported this work.

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