Life Sciences, Vol . 24, pp . Printed in the U.S .A .
1091-1096
Pergamon Press
SUPERORIDE AND HYDROGEN PERORIDE PRODUCTION AND NADPH ORIDATION STIMULATED BY NITROFURANTOIN IN LUNG MICROSOMES : POSSIBLE IMPLICATIONS FOR TORICITY Henry A. Saeame and Michael R . Hoyd* Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, and Clinical Pharmacology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 (Received in final form Fehruarv 9,
1979)
Summary The addition of nitrofurantoin to aerobic incubation miaturea containing rat lung microsomea strongly enhanced the generation of adrenochrome from epinephrine. Adrenochroma for mation in this system was blocked by euperoxide diemutase, but not by catalane . Hydrogen peroxide production vas also strongly enhanced by nitrofurantoin in these preparations ; euperoxide diemutase did not significantly alter the amount of H202 measured, but no H202 was detected in incubation mixtures in the presence Nitrofurantoin enhanced the oxidation of NADPH is of catalane . lung microsomal suspensions under aerobic conditions ; the enhancement was unaffected by catalane but was partially prevented by euperoxide diemutane . Neither adrenochrome formation nor H2O' production were enhanced by nitrofurantoin under anaerobic (N2 conditions, but NADPH oxidation in the preeeace of nitrofurantoin was greater under anaerobic conditions than under These results are consistent with the view aerobic conditions . that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen Species, emphasizing some _in vitro metabolic similarities with the lung-toxic herbicide, paraquat . Mason and Holtzman (1) have pointed out certain similarities in the in vitro pulmonary microsomal metabolism of the widely used antimicrobial drug, nitrofurantoin (N-[5-vitro-2-furfurylidine]-1-aminohydantoin) and the herbi cide, paraquat, which suggest a potential biochemical basin for similar Their studies predicted mechanisms of pulmonary injury by the two agents . that superoxi:de would be produced during the cyclic reduction/oxidation of The generation of eupernitrofurantoin in vitro under aerobic conditions . oxide (2) and hydrogen peroxide (3), and the oxidation of NADPH (3), during the redox cycling of paraquat _in vitro in pulmonary microsomal suspensions have bees previously documented . Pulmonary injury by paraquat has been proposed to result from lipid peroxidation by activated oxygen species formed in the lung during redox cycling of the toxin (4) ; others have suggested that lung injury could result from the disruption of cellular metabolism due to depletion of reduced pyridine nucleotides which might occur during the redox cycling of paraquat (5) .
*Iaquirien and requests for reprints should be addressed to Dr . M.R . Boyd, 20014 . Bldg . 10, Rm . 6N-105, National Cancer Institute, Bethesda, Md .
0024-3205/79/121091-06$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1092
Nitrofurantoin and Pulmonary Injury
Vol . 24, No . 12, 1979
Because of the clinical importance of nitrofurantoia-induced pulmonary disease in man (6), we have characterized further some features of the in vitro pulmonary microsomal metabolism of nitrofurantoin which illustrate several similarities with paraquat . Materiale and Methods Animals and preparation of microsomes . Male, Sprague-Dawley rata (Taconic Farms ; Germantown, N.Y .) weighing 135-140 g were sacrificed by decapitation and the lunge removed and homogenized in 4 vol of 1 .15X KC1-0 .05M Trie buffer, pH 7 .4, and centrifuged at 10,000 x g for 20 minutes; the superThe microsomal natant was removed and centrifuged at 100,000 x g for 1 hour . pallet was resuspeaded in 4 vol of Tria-RC1 and resedimented at 100,000 x g for 1 hour . The washed mícrosomal pellet was resuspended in 0 .05M phosphate buffer, pH 7 .4, prior to use in experiments . Assays were run as described in the footnotes accompanying tables 1-3 . Chemicals and enzymes . Nitrofurantoin, catalase (13000 unite/mg), NADPH, epinephrine, glucose-ó~phosphate, and glucose-ó~phoephate dehydroganase were obtained from Sigma Chemical Co . (St . Louis, Mo .) . Superoxide dismutase (11500 units/mg) was obtained from Miles Laboratories, Ltd . (Cape Town, South All other reagents ware obtained from Fisher Scientific Co . (Silver Africa) . Spring, Md) . Results Earlier _ín vitro studies (1) provided only íadirect evidence that the formation of superoxide was enhanced by nitrofurantoin in lung microsomes under aerobic conditions . Preliminary reports (7,8) suggested that the formation of adrenochrome from epinephrine (9) might provide a more direct indicator of nitrofurantoin-stimulated euperoxide formation . The data in table 1 confirm this view . The formation of adrenochrome was enhanced by nitrofurantoin in incubations with rat lung microsomes in the presence of oxygen, but not under a nitrogen atmosphere . Superoxide dismutase inhibited the nitrofurantoinstimulated adrenochrome formation, confirming that the adrenochrome was derived from the oxidation of epinephrine by superoxide generated in the presence of nitrofurantoin . The dismutase completely prevented adrenochrome formation in the absence of nitrofurantoin . Table 2 indicates that the production of hydrogen peroxide likewise was stimulated by nitrofurantoin in aerobic incubations with rat lung. microsomea . No H202 was detected when incubation mixtures contained catalase, or when the incubations were run under a nitrogen atmosphere . The presence of superoxide dismutase did not alter the total amount of H202 detected after a 10-minute incubation . Nitrofurantoin also markedly enhanced the amount of NADPH oxidized to NADP+ is aerobic incubation mixtures with rat lung mícroeames (table 3) . Under these conditions catalase did not significantly alter the amount of NADP+ formed, but superoxide dismutase caused about a 40X decrease in NADPH oxidation. Under anaerobic conditions the amount of NADP+ formed was actually much greater than under aerobic conditions . Discussion Figure 1 is a schematic representation of some features of the in vitro metabolism of nitrofurantoin which seem to account for the results we have obtained . Mason and Holtzman (1) provided evidence that an anion free-radical (II) of nitrofurantoin was produced in anaerobic incubations of the drug with
Vol . 24, No .
12, 1979
Nitrofurantoin and Pulmonary Injury
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TABLE I Adrenochrome Formation in Rat Lung Microsomes in the Presence of Nitrofurantoin and/or Superoxide Diemutane* Incubation Superoxide Nitrofurantoin Atmosphere Diemuetase (0 .1 mq) (10-3M) +AO.D . x 103 Air 2.6 ± 0 .4 Air + 53 .7 ± 0 .2 Nitrogen + 0 .0 Air + 0 .0 Air + + 14 .1 ± 0 .5 *Adrenochroma vas aesaged by an adaptation of the procedure of Misra and Pridovich (9) . Incubation mixtures contained 1 mg/ml microsomal protein, 10 mM MgC12 and 1 mM epinephrine in a total vol of 3 ml of O .OSM phosphate buffer, pH 7 .4 . Incubations (at 26 .5 °) were started by the addition of 1 ymole of NADPH and the initial rate (within 15 seconds of addition) of +AO.D . 475-b20 mu/min/mg vas recorded using a dual-beam Aminco DW-2 spectrophotometer . Values shown are means t S .E . of triplicate determinations . The incubation with nitrofurantoin under a nitrogen atmosphere initially yielded a baseline with a slightly negative slope, but which rapidly (1 minute) equilibrated to a slope of zero . In aerobic incubations, the addition of nitrofurantoin eignificantlq (P < .O1) enhanced adrenochrome formation ; euperoxide diemutaee significantlq (P < .O1) decreased adrenochrame formation in the presence of the absence of nitrofurantoin . TABLE II Effect of Nitrofurantoin on Hydrogen Peroxide Formation in Rat Lung Microeomee* Conditions
Air atmosphere
Nitrofurantoin (mM) 0.00 0.10 0.25 0.50 1.00 1.00
H202 formed (nmolea/mA/10 minutea~ 14 .0 ± 1 .8 63 .6 ± 9 .6 96 .2 ± 3 .6 128 .4 ± 10 .4 161.1 t 2 .3 0.0
Nitrogen atmosphere Air atmosphere + Superoxide dismutase (0 .2 mg) 1.00 160.4 ± 21 .9 Air atmosphere + Catalans 0.0 (0 .1 mR) 1 .00 *Incubation mixtures contained 1 .5 mg microeomal protein, 3 umoles NADPH, 15 umoles MgC12, and 0 .3 umoles NaNg (omitted with catalane eaperiment) in a Reactions started bq total vol of 1 .5 ml of 0 .05M phosphate buffer, pH 7 .4 . the addition of the NADPH, rue at 37° for 10 minutes, and were stopped by the addition of 1 .0 ml of lOZ HC104 solution . The amounts of H202 in the super natants were assayed colorimetrically according to the method of Hildebraadt, _et al . (10) . Values shown are means ± S .E . of triplicate determinations . Under anaerobic conditions, all concentrations of nitrofurantoin studied caused significant (P < .O1) increases in the amount of H202 formed ; euperoxide dismutaee had no significant effect on the nitrofurantoin-enhanced H202 production .
Nitrofurantoin and Pulmonary Injury
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Vol . 24, No . 12, 1979
TABLE III Effect of Nitrofurantoin on NADPH Oxidation by Rat Lung Microsomes* Conditions
Air atmosphere
Nitrofurantoin (mM) 0 .00 0 .10 0 .25 0 .50
NADP+ formed (nmolea/mg/10 minutes) 5 .0 ± 2 .1 24 .4 t 4 .6 67 .0 ± 4 .0 156 .8 ± 18 .5 245 .2 ± 34 .5 328 .4 t 16 .0
Nitrogen atmosphere 1 .00 Air atmosphere + Superoxide dismutase (0 .1 mg) 1 .0~ 147 .2 ± 14 .4 Air atmosphere + Catalans 1 .00 (0 .1 mg) 195 .9 t 16 .0 *Incubation mixtures contained 1.5 mg of microsomal protein, 15 umoles of MgC1 2 , and an initial concentration of 1 .5 mM tIADPH, in a total vol of 1 .5 ml of 0.05M phosphate buffer, pH 7 .4 . Reactions were started by the addition of the NADPH, run for 10 minutes at 37 ° , and stopped by addition of 1 .0 ml of lOX HC104 . After storage overnight at 4°, 1 .5 ml of 2 M dibasic phosphate solution was added to remove perchlorate from the supernatants . Aliquots (0 .5 ml) of the supernatants were readjusted to pH 7 .4 using 1 N NaOH solution . After the addition of 5 mg of glucose-ó-phosphate to all aliquots, the amounts of NADP+ present were determined by measurements of DO .D . 340-400 mu (dual beam Aminco DW-2 spectrophotometer) before and after the addition of glucose-ó-phosphate dehydrogenase (2 unite) to reduce the NADP+ back to NADPH. Values shown are means t S .E . of triplicate determinations . Superoxide dismutase significantly (P < .O1) decreased the amount of NADP+ formed ; in another experiment, concentrations of superoxide dismutase as low as 5 ug/ml similarly caused about a 40X inhibition of NADP+ formation. Under anaerobic conditions there was significantly (P < .Ol) more NADP+ formed than under aerobic conditions .
lung microsomsn . An anion free-radical metabolite was not detectable in .aerobic incubations . Moreover, the NADPH-dependent uptake of oxygen in pulmonary microsomes was markedly enhanced by the presence of nitrofurantoin under aerobic conditíona ; the net oxygen uptake was decreased by the addition of superoxide dismutase . These results led to the proposal (1) that an anion free-radical metabòlite of nitrofurantoin (II) could react with molecular oxygen to regenerate the parent nitrofurantoin (I) and produce superoxide (fig . 1) . Presumably therefore under aerobic conditions the cyclic reduction/oxidation of nitrofurantoin could generate large amounts of superoxide . Our results are consistent with that view . The adrenochrome studies provided direct evidence for the nitrofurantoin-enhanced superoxide formation (table 1) . Also, as would ba predicted from figure 1, nitrofurantoin enhanced the amounts of H202 formed (table 2) and the amounts of NADPH oxidized to NADP+ in serohic incubations with lung microsomes (table 3) . The latter features are also characteristic of the in vitro metabolism of paraquat (3) . It ís of interest that superoxide dismutase partially prevented the nitrofurantoin-stimulated oxidation of NADPH (table 3) . In other experiments, we found that NADPH was oxidized to NADP+ when added to a superoxide generating system consisting of purified milk xanthine oxidase and hypoxanthine . NADPH oxidation occurred in this system with or without the presence of nitrofurantoin, and was completely prevented
Vol . 24, No . 12, 1979
Nitrofurantoin and Pulmonary Injury
1095
Anavobic Inabttiom Asrobic Ineub~tiau NADP~ NADPH
(NADP~) (NADPHI
2 RNOZ
2 RNOy
[RNOZ]
. [RNO] IIII)
(NADP*) (NADPH)
~[RHNOH]~[RNHZ]
IIVI
IVI
2=0p (Supxoxide Dirnuuw) H202+02 -ìH 2~02 Ghlas
Fig . 1 Schematic representation of some features of the _in vitro metabolism of sitrofuraatoin by addition of superoaida diamutase* . These results suggest the possibility that at least part of the NADP+ formed during aerobic incubations of lung microsomas with nitrofurantoin, or paraquat, could be derived from the direct oxidation of NADPH by an activated oxygen species . It therefore seems plausible that soma of the suparozidn diamutase-sensitive oxygen uptake observed by Mason and Holtzman (1) may have been due to the oxidatioa of NADPH by suparoxide or some product derived therefrom . Nitrofuraatoia also enhanced NADPH oxidation under anaerobic conditioae (table 3) . In the abeeace of oxygen the vitro-reduction of sitrofuraatoin can proceed (fig .l), with the consumption of reducing aquivaleats from NADPH, presumably through the nitroso (III) and hydroxylamino (IV) derivatives and finally to a fully reduced product (V) . Nose of the latter metabolites have bean isolated and conclusively identified from in vitro iacubatioae with lung microsomee, although soma of the reduced products formed in vitro (possibly the hydroxylamino and/or nitroso metabolite) appear to be capable of covalently binding to microeomal macromolecules (11) . Tha covalent binding of nitrofurantoin in vivo does not appear to ba associated with the production of acute pulmonary toxicity in laboratory animals, however, as it is with certain other fume derivatives which do not contain the vitro moiety (12) . Both paraquat and nitrofurantoin can cause severe lung injury is man (6,13) and in laboratory asimale (7,13) . Lung damage is laboratory rodents by both compounds is enhanced by the prior feeding of vitamin É deficient díeta (7,14), or by exposure of tha .animals to oxygen-enriched atmospheres (7,15) . Several pathologic features of acute pulmonary tozicities in rats by the two agente are similar (7,13) . Thus, in vivo studies as wall as the present in vitro results suggest the possibility that aitrofurantoin sad *These results will ba presented in detail in another publication.
1096
Nitrofurantoin and Pulmonary Injury
Vol . 24, No .
12, 1979
As has been proposed paraquat may cause lung damage by similar mechanisms . for paraquat, especially in an oxygen-rich tissue such ae the lung the redox cycling of nitrofurantoin in vivo might yield large amounts of potentially toxic activated oxygen species such ae superoxide [and/or einglet oxygen (4)] and hydrogen peroxide . The redox cycling might also deplete cellular stores of reduced pyridine nucleotides such as NADPH, making the cells yet more vulnerable to oxidant injury . The depletion of NADPH alone seems less likely to be a primary mode of toxicity since nitrofurantoin enhances the _in vitro oxidation of NADPH under both aerobic and anaerobic conditions . Based on the present results, and because of the widespread use of nitrofurantoin, and its potential for causing acute and chronic pulmonary injury in man, we believe that a mechanism of nitrofurantoin-induced lung injury involving oxygen activation merits further investigation . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
R .P . MASON and J .L . HOLT2MAN, Biochem. Biophys . Ree . Commun . 67 12671274 (1975) . M .R . MONTGOMERY, Toxicol . Appl . Pharmacol . 36 543-554 (1976) . K .R . ILETT, B . STRIPP, R .H . MENARD, W.D . REID, and J .R . GILLETTE, Toxicol . Appl . Pharmacol . 28 216-226 (1974) . J .S . BUS, S .D . AUST, and J .E . GIBSON, Biochem . Biophys . Ree . Commun . _58 749-755 (1974) . M.S . ROSE, L .L . SMITH, and I . WYATT, Biochem . Pharmacol . _25 1763-1767 (1976) . E .C . ROSENOW, in Immunologic and Infectious Reactions in the Lung (C .H . Rirkpatrick and H .Y . Reynolds, ade .) pp 261-282, Marcel Dekker, New York (1976) . M . BOYD, H . SASAME, J . MITCHELL, and G . CATIGNANI; Federation Proc . _36 405 (1977) . R .P . MASON, F .J . PETERSON, J .T . CALAGHAN, and J .L . HOLTZMAN, Pharmacologist _19 192 (1977) . H .P . MISRA and I . FRIDOVICH, J . Biol . Chew . _247 3170-3175 (1972) . A .G . HILDEBRANDT and I . ROOTS, Arch . Biochem. Biophys . _171 385-397 (1975) . M .R . BOYD, A.W . STIRO, and H .A . SASAME, Biochem . Pharmacol ., in presa (1979) . M.R . BOYD, Env . Hlth . Perepe . _16 127-138 (1976) . P . SMITH and D . HEATH CRC Crit . Rev . Toxicol . _4 411-445 (1976) . J .S . BUS, S .D . AUST, and J .E . GIBSON, Env . H1th Persps . _16 139-146 (1976) . H .D . FISHER, J .A . CLEMENTS, and R.R . WRIGhT, Am . Rev . Rasp . Dis . _107 246-252 (1973) .
1091-1096
Pergamon Press
SUPERORIDE AND HYDROGEN PERORIDE PRODUCTION AND NADPH ORIDATION STIMULATED BY NITROFURANTOIN IN LUNG MICROSOMES : POSSIBLE IMPLICATIONS FOR TORICITY Henry A. Saeame and Michael R . Hoyd* Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, and Clinical Pharmacology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 (Received in final form Fehruarv 9,
1979)
Summary The addition of nitrofurantoin to aerobic incubation miaturea containing rat lung microsomea strongly enhanced the generation of adrenochrome from epinephrine. Adrenochroma for mation in this system was blocked by euperoxide diemutase, but not by catalane . Hydrogen peroxide production vas also strongly enhanced by nitrofurantoin in these preparations ; euperoxide diemutase did not significantly alter the amount of H202 measured, but no H202 was detected in incubation mixtures in the presence Nitrofurantoin enhanced the oxidation of NADPH is of catalane . lung microsomal suspensions under aerobic conditions ; the enhancement was unaffected by catalane but was partially prevented by euperoxide diemutane . Neither adrenochrome formation nor H2O' production were enhanced by nitrofurantoin under anaerobic (N2 conditions, but NADPH oxidation in the preeeace of nitrofurantoin was greater under anaerobic conditions than under These results are consistent with the view aerobic conditions . that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen Species, emphasizing some _in vitro metabolic similarities with the lung-toxic herbicide, paraquat . Mason and Holtzman (1) have pointed out certain similarities in the in vitro pulmonary microsomal metabolism of the widely used antimicrobial drug, nitrofurantoin (N-[5-vitro-2-furfurylidine]-1-aminohydantoin) and the herbi cide, paraquat, which suggest a potential biochemical basin for similar Their studies predicted mechanisms of pulmonary injury by the two agents . that superoxi:de would be produced during the cyclic reduction/oxidation of The generation of eupernitrofurantoin in vitro under aerobic conditions . oxide (2) and hydrogen peroxide (3), and the oxidation of NADPH (3), during the redox cycling of paraquat _in vitro in pulmonary microsomal suspensions have bees previously documented . Pulmonary injury by paraquat has been proposed to result from lipid peroxidation by activated oxygen species formed in the lung during redox cycling of the toxin (4) ; others have suggested that lung injury could result from the disruption of cellular metabolism due to depletion of reduced pyridine nucleotides which might occur during the redox cycling of paraquat (5) .
*Iaquirien and requests for reprints should be addressed to Dr . M.R . Boyd, 20014 . Bldg . 10, Rm . 6N-105, National Cancer Institute, Bethesda, Md .
0024-3205/79/121091-06$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1092
Nitrofurantoin and Pulmonary Injury
Vol . 24, No . 12, 1979
Because of the clinical importance of nitrofurantoia-induced pulmonary disease in man (6), we have characterized further some features of the in vitro pulmonary microsomal metabolism of nitrofurantoin which illustrate several similarities with paraquat . Materiale and Methods Animals and preparation of microsomes . Male, Sprague-Dawley rata (Taconic Farms ; Germantown, N.Y .) weighing 135-140 g were sacrificed by decapitation and the lunge removed and homogenized in 4 vol of 1 .15X KC1-0 .05M Trie buffer, pH 7 .4, and centrifuged at 10,000 x g for 20 minutes; the superThe microsomal natant was removed and centrifuged at 100,000 x g for 1 hour . pallet was resuspeaded in 4 vol of Tria-RC1 and resedimented at 100,000 x g for 1 hour . The washed mícrosomal pellet was resuspended in 0 .05M phosphate buffer, pH 7 .4, prior to use in experiments . Assays were run as described in the footnotes accompanying tables 1-3 . Chemicals and enzymes . Nitrofurantoin, catalase (13000 unite/mg), NADPH, epinephrine, glucose-ó~phosphate, and glucose-ó~phoephate dehydroganase were obtained from Sigma Chemical Co . (St . Louis, Mo .) . Superoxide dismutase (11500 units/mg) was obtained from Miles Laboratories, Ltd . (Cape Town, South All other reagents ware obtained from Fisher Scientific Co . (Silver Africa) . Spring, Md) . Results Earlier _ín vitro studies (1) provided only íadirect evidence that the formation of superoxide was enhanced by nitrofurantoin in lung microsomes under aerobic conditions . Preliminary reports (7,8) suggested that the formation of adrenochrome from epinephrine (9) might provide a more direct indicator of nitrofurantoin-stimulated euperoxide formation . The data in table 1 confirm this view . The formation of adrenochrome was enhanced by nitrofurantoin in incubations with rat lung microsomes in the presence of oxygen, but not under a nitrogen atmosphere . Superoxide dismutase inhibited the nitrofurantoinstimulated adrenochrome formation, confirming that the adrenochrome was derived from the oxidation of epinephrine by superoxide generated in the presence of nitrofurantoin . The dismutase completely prevented adrenochrome formation in the absence of nitrofurantoin . Table 2 indicates that the production of hydrogen peroxide likewise was stimulated by nitrofurantoin in aerobic incubations with rat lung. microsomea . No H202 was detected when incubation mixtures contained catalase, or when the incubations were run under a nitrogen atmosphere . The presence of superoxide dismutase did not alter the total amount of H202 detected after a 10-minute incubation . Nitrofurantoin also markedly enhanced the amount of NADPH oxidized to NADP+ is aerobic incubation mixtures with rat lung mícroeames (table 3) . Under these conditions catalase did not significantly alter the amount of NADP+ formed, but superoxide dismutase caused about a 40X decrease in NADPH oxidation. Under anaerobic conditions the amount of NADP+ formed was actually much greater than under aerobic conditions . Discussion Figure 1 is a schematic representation of some features of the in vitro metabolism of nitrofurantoin which seem to account for the results we have obtained . Mason and Holtzman (1) provided evidence that an anion free-radical (II) of nitrofurantoin was produced in anaerobic incubations of the drug with
Vol . 24, No .
12, 1979
Nitrofurantoin and Pulmonary Injury
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TABLE I Adrenochrome Formation in Rat Lung Microsomes in the Presence of Nitrofurantoin and/or Superoxide Diemutane* Incubation Superoxide Nitrofurantoin Atmosphere Diemuetase (0 .1 mq) (10-3M) +AO.D . x 103 Air 2.6 ± 0 .4 Air + 53 .7 ± 0 .2 Nitrogen + 0 .0 Air + 0 .0 Air + + 14 .1 ± 0 .5 *Adrenochroma vas aesaged by an adaptation of the procedure of Misra and Pridovich (9) . Incubation mixtures contained 1 mg/ml microsomal protein, 10 mM MgC12 and 1 mM epinephrine in a total vol of 3 ml of O .OSM phosphate buffer, pH 7 .4 . Incubations (at 26 .5 °) were started by the addition of 1 ymole of NADPH and the initial rate (within 15 seconds of addition) of +AO.D . 475-b20 mu/min/mg vas recorded using a dual-beam Aminco DW-2 spectrophotometer . Values shown are means t S .E . of triplicate determinations . The incubation with nitrofurantoin under a nitrogen atmosphere initially yielded a baseline with a slightly negative slope, but which rapidly (1 minute) equilibrated to a slope of zero . In aerobic incubations, the addition of nitrofurantoin eignificantlq (P < .O1) enhanced adrenochrome formation ; euperoxide diemutaee significantlq (P < .O1) decreased adrenochrame formation in the presence of the absence of nitrofurantoin . TABLE II Effect of Nitrofurantoin on Hydrogen Peroxide Formation in Rat Lung Microeomee* Conditions
Air atmosphere
Nitrofurantoin (mM) 0.00 0.10 0.25 0.50 1.00 1.00
H202 formed (nmolea/mA/10 minutea~ 14 .0 ± 1 .8 63 .6 ± 9 .6 96 .2 ± 3 .6 128 .4 ± 10 .4 161.1 t 2 .3 0.0
Nitrogen atmosphere Air atmosphere + Superoxide dismutase (0 .2 mg) 1.00 160.4 ± 21 .9 Air atmosphere + Catalans 0.0 (0 .1 mR) 1 .00 *Incubation mixtures contained 1 .5 mg microeomal protein, 3 umoles NADPH, 15 umoles MgC12, and 0 .3 umoles NaNg (omitted with catalane eaperiment) in a Reactions started bq total vol of 1 .5 ml of 0 .05M phosphate buffer, pH 7 .4 . the addition of the NADPH, rue at 37° for 10 minutes, and were stopped by the addition of 1 .0 ml of lOZ HC104 solution . The amounts of H202 in the super natants were assayed colorimetrically according to the method of Hildebraadt, _et al . (10) . Values shown are means ± S .E . of triplicate determinations . Under anaerobic conditions, all concentrations of nitrofurantoin studied caused significant (P < .O1) increases in the amount of H202 formed ; euperoxide dismutaee had no significant effect on the nitrofurantoin-enhanced H202 production .
Nitrofurantoin and Pulmonary Injury
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Vol . 24, No . 12, 1979
TABLE III Effect of Nitrofurantoin on NADPH Oxidation by Rat Lung Microsomes* Conditions
Air atmosphere
Nitrofurantoin (mM) 0 .00 0 .10 0 .25 0 .50
NADP+ formed (nmolea/mg/10 minutes) 5 .0 ± 2 .1 24 .4 t 4 .6 67 .0 ± 4 .0 156 .8 ± 18 .5 245 .2 ± 34 .5 328 .4 t 16 .0
Nitrogen atmosphere 1 .00 Air atmosphere + Superoxide dismutase (0 .1 mg) 1 .0~ 147 .2 ± 14 .4 Air atmosphere + Catalans 1 .00 (0 .1 mg) 195 .9 t 16 .0 *Incubation mixtures contained 1.5 mg of microsomal protein, 15 umoles of MgC1 2 , and an initial concentration of 1 .5 mM tIADPH, in a total vol of 1 .5 ml of 0.05M phosphate buffer, pH 7 .4 . Reactions were started by the addition of the NADPH, run for 10 minutes at 37 ° , and stopped by addition of 1 .0 ml of lOX HC104 . After storage overnight at 4°, 1 .5 ml of 2 M dibasic phosphate solution was added to remove perchlorate from the supernatants . Aliquots (0 .5 ml) of the supernatants were readjusted to pH 7 .4 using 1 N NaOH solution . After the addition of 5 mg of glucose-ó-phosphate to all aliquots, the amounts of NADP+ present were determined by measurements of DO .D . 340-400 mu (dual beam Aminco DW-2 spectrophotometer) before and after the addition of glucose-ó-phosphate dehydrogenase (2 unite) to reduce the NADP+ back to NADPH. Values shown are means t S .E . of triplicate determinations . Superoxide dismutase significantly (P < .O1) decreased the amount of NADP+ formed ; in another experiment, concentrations of superoxide dismutase as low as 5 ug/ml similarly caused about a 40X inhibition of NADP+ formation. Under anaerobic conditions there was significantly (P < .Ol) more NADP+ formed than under aerobic conditions .
lung microsomsn . An anion free-radical metabolite was not detectable in .aerobic incubations . Moreover, the NADPH-dependent uptake of oxygen in pulmonary microsomes was markedly enhanced by the presence of nitrofurantoin under aerobic conditíona ; the net oxygen uptake was decreased by the addition of superoxide dismutase . These results led to the proposal (1) that an anion free-radical metabòlite of nitrofurantoin (II) could react with molecular oxygen to regenerate the parent nitrofurantoin (I) and produce superoxide (fig . 1) . Presumably therefore under aerobic conditions the cyclic reduction/oxidation of nitrofurantoin could generate large amounts of superoxide . Our results are consistent with that view . The adrenochrome studies provided direct evidence for the nitrofurantoin-enhanced superoxide formation (table 1) . Also, as would ba predicted from figure 1, nitrofurantoin enhanced the amounts of H202 formed (table 2) and the amounts of NADPH oxidized to NADP+ in serohic incubations with lung microsomes (table 3) . The latter features are also characteristic of the in vitro metabolism of paraquat (3) . It ís of interest that superoxide dismutase partially prevented the nitrofurantoin-stimulated oxidation of NADPH (table 3) . In other experiments, we found that NADPH was oxidized to NADP+ when added to a superoxide generating system consisting of purified milk xanthine oxidase and hypoxanthine . NADPH oxidation occurred in this system with or without the presence of nitrofurantoin, and was completely prevented
Vol . 24, No . 12, 1979
Nitrofurantoin and Pulmonary Injury
1095
Anavobic Inabttiom Asrobic Ineub~tiau NADP~ NADPH
(NADP~) (NADPHI
2 RNOZ
2 RNOy
[RNOZ]
. [RNO] IIII)
(NADP*) (NADPH)
~[RHNOH]~[RNHZ]
IIVI
IVI
2=0p (Supxoxide Dirnuuw) H202+02 -ìH 2~02 Ghlas
Fig . 1 Schematic representation of some features of the _in vitro metabolism of sitrofuraatoin by addition of superoaida diamutase* . These results suggest the possibility that at least part of the NADP+ formed during aerobic incubations of lung microsomas with nitrofurantoin, or paraquat, could be derived from the direct oxidation of NADPH by an activated oxygen species . It therefore seems plausible that soma of the suparozidn diamutase-sensitive oxygen uptake observed by Mason and Holtzman (1) may have been due to the oxidatioa of NADPH by suparoxide or some product derived therefrom . Nitrofuraatoia also enhanced NADPH oxidation under anaerobic conditioae (table 3) . In the abeeace of oxygen the vitro-reduction of sitrofuraatoin can proceed (fig .l), with the consumption of reducing aquivaleats from NADPH, presumably through the nitroso (III) and hydroxylamino (IV) derivatives and finally to a fully reduced product (V) . Nose of the latter metabolites have bean isolated and conclusively identified from in vitro iacubatioae with lung microsomee, although soma of the reduced products formed in vitro (possibly the hydroxylamino and/or nitroso metabolite) appear to be capable of covalently binding to microeomal macromolecules (11) . Tha covalent binding of nitrofurantoin in vivo does not appear to ba associated with the production of acute pulmonary toxicity in laboratory animals, however, as it is with certain other fume derivatives which do not contain the vitro moiety (12) . Both paraquat and nitrofurantoin can cause severe lung injury is man (6,13) and in laboratory asimale (7,13) . Lung damage is laboratory rodents by both compounds is enhanced by the prior feeding of vitamin É deficient díeta (7,14), or by exposure of tha .animals to oxygen-enriched atmospheres (7,15) . Several pathologic features of acute pulmonary tozicities in rats by the two agente are similar (7,13) . Thus, in vivo studies as wall as the present in vitro results suggest the possibility that aitrofurantoin sad *These results will ba presented in detail in another publication.
1096
Nitrofurantoin and Pulmonary Injury
Vol . 24, No .
12, 1979
As has been proposed paraquat may cause lung damage by similar mechanisms . for paraquat, especially in an oxygen-rich tissue such ae the lung the redox cycling of nitrofurantoin in vivo might yield large amounts of potentially toxic activated oxygen species such ae superoxide [and/or einglet oxygen (4)] and hydrogen peroxide . The redox cycling might also deplete cellular stores of reduced pyridine nucleotides such as NADPH, making the cells yet more vulnerable to oxidant injury . The depletion of NADPH alone seems less likely to be a primary mode of toxicity since nitrofurantoin enhances the _in vitro oxidation of NADPH under both aerobic and anaerobic conditions . Based on the present results, and because of the widespread use of nitrofurantoin, and its potential for causing acute and chronic pulmonary injury in man, we believe that a mechanism of nitrofurantoin-induced lung injury involving oxygen activation merits further investigation . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
R .P . MASON and J .L . HOLT2MAN, Biochem. Biophys . Ree . Commun . 67 12671274 (1975) . M .R . MONTGOMERY, Toxicol . Appl . Pharmacol . 36 543-554 (1976) . K .R . ILETT, B . STRIPP, R .H . MENARD, W.D . REID, and J .R . GILLETTE, Toxicol . Appl . Pharmacol . 28 216-226 (1974) . J .S . BUS, S .D . AUST, and J .E . GIBSON, Biochem . Biophys . Ree . Commun . _58 749-755 (1974) . M.S . ROSE, L .L . SMITH, and I . WYATT, Biochem . Pharmacol . _25 1763-1767 (1976) . E .C . ROSENOW, in Immunologic and Infectious Reactions in the Lung (C .H . Rirkpatrick and H .Y . Reynolds, ade .) pp 261-282, Marcel Dekker, New York (1976) . M . BOYD, H . SASAME, J . MITCHELL, and G . CATIGNANI; Federation Proc . _36 405 (1977) . R .P . MASON, F .J . PETERSON, J .T . CALAGHAN, and J .L . HOLTZMAN, Pharmacologist _19 192 (1977) . H .P . MISRA and I . FRIDOVICH, J . Biol . Chew . _247 3170-3175 (1972) . A .G . HILDEBRANDT and I . ROOTS, Arch . Biochem. Biophys . _171 385-397 (1975) . M .R . BOYD, A.W . STIRO, and H .A . SASAME, Biochem . Pharmacol ., in presa (1979) . M.R . BOYD, Env . Hlth . Perepe . _16 127-138 (1976) . P . SMITH and D . HEATH CRC Crit . Rev . Toxicol . _4 411-445 (1976) . J .S . BUS, S .D . AUST, and J .E . GIBSON, Env . H1th Persps . _16 139-146 (1976) . H .D . FISHER, J .A . CLEMENTS, and R.R . WRIGhT, Am . Rev . Rasp . Dis . _107 246-252 (1973) .
