Revisiting the Interaction of the Radical Anion Metabolite of 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 Environmental Health 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 the scavenging nature of glutathione toward the nitro radical anion of the drug nitrofurantoin. We produced the radical anion enzymatically using the xanthine oxidase/hypoxanthine system at pH 7.4 and pH 9.0 in the presence of various levels of glutathione from 10 to 100 mM and monitored any changes in the radical concentration via electron spin resonance spectros- copy. Independent of glutathione concentration, there was no decrease in the steady-state concentration of the radical. In fact, there was an average 30% increase in 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 the radical 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; stopped flow. Nitrofurantoin (N-(5-nitro-2-furfuryldine)-1-amino- hydantoin) (NFT) 2 is a nitroaromatic compound that is used as a urinary antimicrobial agent (1, 2). It belongs to the class of nitroheterocyclic compounds, which in- cludes nitrofurans and nitroimidazoles. These com- pounds been investigated for use in cancer therapy as radiosensitizers and hypoxia-specific cytotoxic agents (3–7). However, these compounds have been associated with lung and liver damage (2). The mechanism(s) of cytotoxicity is not well understood, except that the nitro radical anion is an obligate intermediate, and chemical damage may be linked to its further reduction products 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 biochemical reducing agents such as ascorbate and catecholamines (3, 5, 8). ArNO 2 O ¡ 1e– enzyme ArNO 2 •- [1] Once formed, the nitro anion radical disproportionates under 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 reduces oxygen to the superoxide radical anion, O 2 •- (9 –11). ArNO 2 •- + O 2 3 ArNO 2 + O 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). 2ArNO 2 •- + 2H + 3 ArNO 2 + ArNO + H 2 O [3] 1 To whom correspondence and reprint requests should be ad- dressed at Department of Chemistry, John Carroll University, 20700 North 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 $35.00 113 © 2001 Elsevier Science All rights reserved. Archives of Biochemistry and Biophysics Vol. 397, No. 1, January 1, pp. 113–118, 2002 doi:10.1006/abbi.2001.2670, available online at http://www.idealibrary.com on