Reduction of nifurtimox and nitrofurantoin to free radical metabolites by rat liver mitochondria. Evidence of an outer membrane-located nitroreductase.

S N Moreno,R P Mason,R Docampo

doi:10.1016/s0021-9258(20)82140-1

Abstract

Nifurtimox and nitrofurantoin are reduced by intact rat liver mitochondria to nitro anion radicals whose autoxidation generates superoxide anion as detected by direct electron spin resonance spectroscopy and by spin-trapping experiments, respectively. Although nitroreduction occurred in the presence of respiratory substrates such as beta-hydroxybutyrate, malate-glutamate, succinate, or endogenous substrates, nitro anion radical formation activity was much greater on addition of exogenous reduced pyridine nucleotides. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H nitroreductase reactions unless the mitochondria were solubilized by detergent. In addition, NAD(P)H nitroreductase activity was detected in the crude mitochondrial outer membrane fraction, with a higher activity than in mitoplasts and intact mitochondria. These results provide direct evidence of a nitrofuran reductase activity associated with the mitochondrial outer membrane that is far more important than that of respiratory chain enzymes.

Highlights

Nifurtimox and nitrofurantoinare reduced by intact concentrations of nifurtimox are able to induce maximal rat Iiver mitochondria to nitro anion radicals whose stimulation of 0,production by Trypanosomacruzi mitochondrial fraction and to initiate by direct electron spin resonance spectroscopy and by diffusion ofH,O, outside the cells [7, 8]
Ni- with rat liver microsomeshave suggested that nifurtimox [9], troreduction occurred in the presence of respiratory like nitrofurantoin, is rapidly reduced in the presence of substratessuch as B-hydroxybutyrate,malate-gluta- pyridine nucleotides to the nitro anion radical which, in the mate, succinate,or endogenous substrates, nitro anionpresence of air, reacts readily with oxygen to form superoxide radical formationactivity was much greater on addi- and to regenerate the parent compound [11].These findings tioonefxogenourseducepdyridinneucleotides
The incubation of nifurtimox or nitrofurantoin with rat liver mitochondriain the presence of mitochondrialsubstrates such as succinate or 8-hydroxybutyrate generates multiline

Summary

THEJOURNAL OF BIOLOGICACLHEMISTRY

Vol 259, No 10, Issue of May 25, pp. 6298+5,1984 Printed u1 U.S.A. Reduction of Nifurtimox and Nitrofurantoin to Free Radical Metabolites by RatLiver Mitochondria. In ad- Nitrofurans are known to be reduced in vitro by cytosol dition, NAD(P)H nitroreductaseactivity was detected enzymes such as aldehyde oxidase [12]and xanthine oxidase in the crude mitochondrial outer membrane fraction, [13] or by flavin-containing microsomalenzymes such as with a higher activity than in mitoplasts and intact cytochrome c reductases [14]. Most 5-nitrofurans, when adequately determined that nifurtimox and nitrofurantoin are enzymattested, have been shown to have mutagenic, cytotoxic, and ically converted to nitro anion radicals capable of participatcarcinogenic activities [4]. Spin-trapping experiments were carried out asdescribed before [34] in thesame incubation medium described above containing 100 mM DMPO.' DMPO was purified by fractional vaccum distiIlation [35]. Cytochrome P-450 was measured in an Aminco DW-2a spectrophotometer according to theprocedure of Omura and Sat0(38)

RESULTS

Mitochondria Microsomes Supernatant nmolfmin mg protein nmolfmin mg protein

None NADH

Reduction of Nitrofuranbsy Rat LiverMitochondria