Generation of furazolidone radical anion and its inhibition by glutathione

BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

48,

56-63

(1992)

Generation of Furazolidone Radical Anion and Its Inhibition by Glutathione DANIELA Departments

of *Pediatrics

LAX*

AND STEPHEN G. KUKOLICH~

and the Children’s Research Arizona, Tucson, Arizona

Center and tchemistry, 85724

University

of

Received March 30. 1992 Furazolidone is a nitrofuran drug which causes dilated cardiomyopathy in turkeys and serves as an important model of human dilated cardiomyopathy. Although extensively investigated, the chemical mechanism by which furazolidone produces injury remains unknown. In this work we used electron paramagnetic resonance (EPR) spectroscopy to show that furazolidone was reduced to its corresponding nitro anion radical by ascorbate and hypoxanthine. Glutathione prevented the generation of this anion radical. These results document directly, with EPR spectroscopy, the presence of furazolidone anion radical during biochemical reduction and suggest a protective role of glutathione in furazolidone-induced injury. These data enhance our understanding of furazolidone metabolism and may be useful in defining its role in furazolidone-induced dilated cardiomyopathy. o 199~ Academic press. I~C.

Furazolidone is a nitrofuran antibiotic which causes dilated cardiomyopathy in turkeys, ducklings and chicks (l-3). The drug induced cardiomyopathy in turkeys serves as an excellent model for dilated cardiomyopathy in humans. Although extensively investigated, the exact mechanism of furazolidone induced injury is undetermined (4-S). Free radicals have been shown to cause direct myocardial injury (9) and evidence suggests that free radical interactions may be implicated in furazolidone-induced damage (10). The major route of nitro drug metabolism is reduction to the corresponding nitro anion radical; it is this reactive intermediate which is thought to mediate many of the nitrofuran-associated toxicities (11,12). Furazolidone has also been reported to undergo nitro reduction and the presence of several metabolites and conjugates has been documented indirectly using nuclear magnetic resonance spectroscopy (NMR) and high pressure liquid chromatography (HPLC) (13-15). Electron paramagnetic resonance (EPR) spectroscopy, however, is necessary to document directly the presence of free radical intermediates generated during furazolidone metabolism. Nitro anion radicals of nitroaromatic compounds other than furazolidone (nitrofurantoin, misonidazole, metronidazole) have been generated by ascorbate (16) and hypoxanthine and xanthine oxidase (12,17); the presence of these nitro anion radicals was documented with EPR spectroscopy. 56 08854505192

$5.00

Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

GENERATION

OF FURAZOLIDONE

RADICAL

57

We report the reduction of furazolidone to its anion radical by ascorbate and hypoxanthine and document the presence of this reactive intermediate directly with EPR spectroscopy. In addition, we show that glutathione inhibits the formation of furazolidone anion radical intermediate. MATERIALS AND METHODS Sodium ascorbate, dimethylformamide, sodium hydroxide, glutathione, hypoxanthine and xanthine oxidase, and Hepes buffer were obtained from Sigma Chemical Company (St. Louis, MO); furazolidone was purchased from Aldrich Chemical Company. EPR measurements were performed at room temperature with Varian E-3 spectrometer operating near 9.2 GHz; instrument conditions were as indicated in figures. Dimethylformamide was used as a solvent for furazolidone in order to maintain final reaction mixture volume at 3 ml. Sodium ascorbate was dissolved in 0.1 N NaOH under a stream of nitrogen. For the experiment illustrated in Fig. 1, 0.3 ml of 100 mM sodium ascorbate solution (in NaOH) was used; in Fig. 4, 0.033 ml of 100 nu+# sodium ascorbate solution was used with 2.967 ml DMF to maintain final volume 3 ml. Glutathione was dissolved in 1.25 N NaOH to maintain stable, alkaline pH of reaction mixture. Xanthine oxidase was diluted in Hepes buffer at a final concentration of 0.86 units/ml. Hypoxanthine was dissolved in 10 N NaOH and Hepes buffer, pH 7.6, under nitrogen, for final hypoxanthine solution pH 14.5. Final concentrations were 9 m&f furazolidone, 10 mM sodium ascorbate, 100, 50,25, and 5 mM glutathione, 3.2 mM hypoxanthine, 0.86 units/ml xanthine oxidase. All solutions were freshly prepared, added together in a borosilicate glass tube, immediately transferred to an EPR quartz flat cell (Wilmad Glass Company Inc., Buena, NJ) using a borosilicate glass pipet, and the EPR spectrum was recorded in the 3400 G region (near g = 2). The assignments of hyperfine splitting constants to various nuclei in the molecule are based on previous work on nitrofurantoin (16). The hyperfine splitting of the electron spin energy levels is described by the first-order energy shifts AE(mi, m,) = C aimims, where ai are the hyperfine coupling constants due to nitrogen (aN) or hydrogen (a”) nuclei, mi are the nuclear spin quantum numbers, and m, the electron spin magnetic quantum number. Simulated first-derivative spectra were computer generated using estimated values for the hyperhne coupling constants and the values of these hyperfine coupling constants, ai, were adjusted to produce a good match between the experimental and simulated spectrum. Three nitrogen and three hydrogen hyperfine coupling constants in the furazolidone molecule were determined by this procedure. The two smallest hydrogen hyperfine constants aH2 and aH3 were obtained directly from the outermost three lines of the high resolution spectrum. Ascorbate radical, g-value, g(ascorbate) = 2.0052 (18) was used as a reference to calibrate our spectra. RESULTS Furazolidone anion radicals were generated by reducing furazolidone using basic ascorbate solution, hypoxanthine, or a hypoxanthine/xanthine oxidase combi-

58

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AND

KUKOLICH

nation. The EPR spectrum obtained using ascorbate is shown in Fig. 1. In addition to the multiline spectrum assigned to the furazolidone radicals, two doublets were seen near the center which are due to the ascorbate radical. The furazolidone anion radical spectrum obtained using basic hypoxanthine is shown in Fig. 2. This is higher amplitude but otherwise identical to the furazolidone radical spectrum in Fig. 1 and to the spectrum obtained using hypoxanthine and xanthine oxidase (not shown). The best-resolved spectra show approximately 70 lines using 0.1 G modulation, such that some overmodulated spectra were taken to aid in the analysis. Spectra taken with 0.8 and 1.25 G modulation proved most useful in the analysis. An example of the comparison of experimental and computer simulated spectra for 1.25 G modulation is shown in Fig. 3. Our values of the hyperfine coupling constants are shown in Table 1. Comparisons of hyperfine coupling constants in the present work to furazolidone anion radical generated by electrochemical methods (19) and to nitrofurantoin anion radical (16) are made in the table. The center of the furazolidone spectrum was approximately 1 G upfield from the center of the ascorbate radical doublet. The g-value and hyperfine constants for ascorbate radical are given by Laroff et al. (18) and their reported g-value, g(ascorbate) = 2.0052, is used as a reference to calibrate our spectra. The furazolidone radical g-value obtained using this calibration is g(furazolidone) = 2.0049 (3).

Glutathione completely inhibited the production of furazolidone anion radical when generated by either ascorbate or hypoxanthine Figs. 1 and 2, lines 4 and 3, respectively. Glutathione also inhibited the generation of ascorbate radical (Fig. 4). Since glutathione at high concentrations may acidify reactions and thus enhance nitro anion radical decay, care was taken to maintain stable alkaline pH of the reaction mixture. Minimal decrease (14.95 to 14.55) in pH was detected after addition of glutathione. In order to generate furazolidone anion radical, it was necessary to prepare ascorbate and hypoxanthine anaerobically; however, the addition of reagents to furazolidone was performed aerobically or anaerobically under argon. Nitro anion radical was detected during aerobic or anaerobic addition of reagents. The furazolidone and ascorbate reaction mixture was extremely alkaline at pH 14. Similar pH was present in other reaction mixtures containing hypoxanthine and glutathione. DISCUSSION The results of this study document the generation of nitro anion radical during furazolidone metabolism directly with EPR spectroscopy. To our knowledge this is the first EPR study of furazolidone anion radical produced by ascorbate or biochemical reduction. Anion radicals of nitro compounds can be produced biochemically with hypoxanthine and xanthine oxidase as well as with ascorbate. Rao et al. (16) showed that nitrofurantoin, misonidazole, and metronidazole were reduced to their corresponding nitro anion radical by ascorbate, thus explaining in part the role of ascorbate in enhancing nitro compound toxicity in hypoxic cells. Mason and Josephy (12) demonstrated nitro anion radical generation of

GENERATION

OF FURAZOLIDONE

RADICAL

59

2.

FIG. 1. EPR spectrum of furazolidone anion radical generated by ascorbate and its inhibition by glutathione is shown. Lines 1 and 2 are controls with furazolidone (9 mM in 1.35 ml dimethylformamide) and Na ascorbate (10 m&f in 0.1 N NaOH, 0.3 ml prepared anaerobically, and 1.35 ml dimethylformamide) alone, respectively; no free radical species is demonstrated. Line 3 is an EPR spectrum of furazolidone anion radical produced in a system of 9 mM furazolidone and 10 mM sodium ascorbate in 0.1 N NaOH (prepared anaerobically), final pH 14.95; the 2 doublets seen near the center are due to ascorbate radical. Line 4 illustrates complete inhibition of free radical generation by 250 mM glutathione final pH 14.55. Line 5 is a lower amplitude furazolidone anion radical, consistent with partial inhibition of free radical generation with 25 mM glutathione. Instrumental conditions were 10 mW microwave power, 0.1 G modulation amplitude, 0.3 s time constant, 9.52 GHz microwave frequency, and 3395-3397 G field set.

misonidazole following incubation with hypoxanthine and xanthine oxidase. Our data show that furazolidone anion radical is generated after incubation with both ascorbate as in the work of Rao et al. (16) and hypoxanthine with or without xanthine oxidase. The ability to generate furazolidone anion radical in the absence of xanthine oxidase is consistent with the work of Mason and Josephy (12) who noted that hypoxanthine is the ultimate source of the extra electron. The only other report in the literature of furazolidone anion radical documentation with EPR spectroscopy is by Gavars et al. (19) who used nonbiochemical techniques (electrochemistry) to generate furazolidone nitro anion radical. Our values of the hyperfine coupling constants shown in Table 1 are similar to those obtained by Gavars et al. (19). The slight differences between our work and that of Gavars et al. (18) and Rao et al. (16) could be, in part, due to different solvents: DMF only for Gavars et al. compared to a combination of DMF and aqueous in the present work and aqueous for nitrofurantoin (16). In addition, nitrofurantoin differs structurally from furazolidone and a slightly different spectrum would be expected. Our results show that generation of a radical intermediate occurred at high pH (~13); this is consistent with the work of Morales et al. (20) who studied the voltammetric behavior of furazolidone and found that at pH 4.5 furazolidone was reduced in a single six-electron wave but at very high pH (>S.Sj a different

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FIG. 2. EPR spectrum of furazolidone anion radical generated by hypoxanthine and its inhibition by glutathione is illustrated. Line 1 is control, hypoxanthine alone (3.2 mM in NaOH), no free radical species is demonstrated. Line 2 shows the EPR spectrum of furazolidone anion radical produced in a system of 9 mM furazolidone and 3.2 mM hypoxanthine (prepared anaerobically). Line 3 shows complete inhibition of furazolidone anion radical by a25 m&f glutathione. Line 4 shows a lower amplitude furazolidone anion radical, consistent with partial inhibition of free radical generation by 5 m&f glutathione. Instrumental conditions were 10 mW microwave power, 0.1 G modulation amplitude, 0.3 s time constant, 9.52 GHz microwave frequency, and 3395 G field set.

8

I

3375

3380

3385

3390

3395

&400

3405

3410

3415

3420

3425

FIG. 3. Overmodulated (----) and computer simulations (-) of the EPR spectrum of furazolidone anion radical are illustrated. Experimental conditions were 9 mM furazolidone and 3.2 mM hypoxanthine as described in the legend for Fig. 2, line 2. Instrumental conditions were 10 mW microwave power, 1.25 G modulation amplitude, 0.3 s time constant, 9.52 GHz microwave frequency, and 3395 G field set.

GENERATION

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RADICAL

TABLE 1 Hypertine Coupling Constants a, for Nitrogen and Hydrogen (in gauss) Furazolidone Present work aNI (NO4 aNZW=N) aN3 k-N=) aHI (H4) aHZ(H3) am (H(==N)

8.8 2.5 0.59 5.2 1.3 0.97

Gavars et

(6) (4) (8) (8) (6) (8)

al.

7.00 1.98 0.40 4.35 1.19 0.89

(19)

Nitrofurantoin (Rao et al. (16)) 11.03 2.30 0.31 5.91 1.53 1.0

process occurred, involving reversible reduction of nitro group to a nitro anion radical. Nitro anion radical was not demonstrated using EPR spectroscopy in their work. Other investigators also documented that the decay of nitro anion radicals is acid catalyzed (21,22). Furazolidone metabolism has been previously investigated. Abraham et al. (13) and Vroomen et al. (14,159 used NMR and HPLC to document the presence of a conjugate with glutathione or mercaptoethanol and suggested that in order for this conjugate to have formed, a reactive intermediate must have combined with glutathione or mercaptoethanol. The fact that in the present work no furazolidone anion radical was detected following addition of glutathione could suggest the formation of such a glutathione/furazolidone conjugate; however, the specific

L ---A FIG. 4. EPR spectrum of ascorbate radical and its inhibition by glutathione is illustrated. Top line indicates ascorbate radical produced in a system of 1.1 n&f sodium ascorbate in 0.1 N NaOH (0.033 ml, prepared anaerobically) and dimethylformamide (2.967 ml). Bottom line shows inhibition of ascorbate radical by glutathione in a system of 1.1 mM sodium ascorbate + 1.1 mM glutathione and dimethylformamide. Instrumental conditions were 10 mW microwave power, 0.16 G modulation amplitude, 1.0 s time constant, 9.52 GHz microwave frequency, and 3397 G field set.

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mechanism by which glutathione inhibits the generation of furazolidone anion radical was not addressed. Furazolidone metabolism is similar to that of other nitro drugs such as nitrofurantoin in that it can be reduced to a nitro anion radical; however, furazolidone anion radical is different from nitrofurantoin anion radical in that glutathione did not inhibit the generation of an anion free radical metabolite of nitrofurantoin (23). Polnaszek et al. (23) investigated the reaction of nitro anion radical of N[4-(5nitro-2-furyl)-2-thiazolyllacetamide (NFIA) and nitrofurantoin with glutathione and found that the steady-state EPR amplitude of the anion radical of both compounds was not affected by glutathione even at high concentrations of 100 m&f. Our results clearly demonstrate the inhibition of reactive intermediate following the addition of glutathione at 225 mM concentration to either furazolidone/ascorbate or furazolidone/hypoxanthine systems. The reason why furazolidone anion radical behaves differently from nitrofurantoin is as yet undetermined; however, it is significant since glutathione may serve as a protective agent in furazolidone-induced injury. Our results document directly the presence of anion radical during biochemical reduction of furazolidone and confirm the similarity of furazolidone metabolism to other nitro drugs. In contrast to other nitro compounds, the generation of furazolidone anion radical is inhibited with glutathione. These data enhance our understanding of furazolidone metabolism and will be useful in defining its role in furazolidone induced cardiac injury. ACKNOWLEDGMENTS This study was supported by the American Heart Association, Arizona Affiliate Grant IG-2-29-90. The results were presented, in part, at the Society for Pediatric Research meeting in New Orleans, Louisiana, May 1991.

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