Structural differences between hypoxanthine phosphoribosyltransferase family members highlight opportunities for antiparasitic drug design in neglected diseases.

Approaches to identify novel druggable targets for treating neglected diseases include computational studies that predict possible interactions of drugs and their molecular targets. Hypoxanthine phosphoribosyltransferase (HPRT) plays a central role in the purine salvage pathway. This enzyme is essential for the survival of the protozoan parasite T. cruzi, the causal agent of Chagas disease, and other parasites related to neglected diseases. Here we found dissimilar functional behaviours between TcHPRT and the human homologue, HsHPRT, in the presence of substrate analogues that can lie in differences in their oligomeric assemblies and structural features. To shed light on this issue, we carried out a comparative structural analysis between both enzymes. Our results show that HsHPRT is considerably more resistant to controlled proteolysis than TcHPRT. Moreover, we observed a variation in the length of two key loops depending on the structural arrangement of each protein (groups D1T1 and D1T1'). Such variations might be involved in inter-subunit communication or influencing the oligomeric state. Besides, to understand the molecular basis that govern D1T1 and D1T1' folding groups, we explored the distribution of charges on the interaction surfaces of TcHPRT and HsHPRT, respectively. To know whether the rigidity degree bears effect on the active site, we studied the flexibility of both proteins. The analysis performed here illuminates the underlying reasons and significance behind each protein's preference for one or the other quaternary arrangement that can be exploited for therapeutic approaches.

Zoledronate repositioning as a potential trypanocidal drug. Trypanosoma cruzi HPRT an alternative target to be considered.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 7 million people worldwide. Considering the side effects and drug resistance shown by current treatments, the development of new anti-Chagas therapies is an urgent need. T. cruzi hypoxanthine phosphoribosyltransferase (TcHPRT), the key enzyme of the purine salvage pathway, is essential for the survival of trypanosomatids. Previously, we assessed the inhibitory effect of different bisphosphonates (BPs), HPRT substrate analogues, on the activity of the isolated enzyme. BPs are used as a treatment for bone diseases and growth inhibition studies on T. cruzi have associated BPs action with the farnesyl diphosphate synthase inhibition. Here, we demonstrated significant growth inhibition of epimastigotes in the presence of BPs and a strong correlation with our previous results on the isolated TcHPRT, suggesting this enzyme as a possible and important target for these drugs. We also found that the parasites exhibited a delay at S phase in the presence of zoledronate pointing out enzymes involved in the cell cycle, such as TcHPRT, as intracellular targets. Moreover, we validated that micromolar concentrations of zoledronate are capable to interfere with the progression of cell infection by this parasite. Altogether, our findings allow us to propose the repositioning of zoledronate as a promising candidate against Chagas disease and TcHPRT as a new target for future rational design of antiparasitic drugs.

[Inhibitory action of Fenton systems on topoisomerase I from Trypanosoma cruzi and Crithidia fasciculata]]. / Acción inhibitoria de sistemas Fenton sobre la topoisomerasa I de Trypanosoma cruzi y Crithidia fasciculata.

Fenton systems (H2O2/Fe(II) or H2O2/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 mM Fe(II) or Cu(II) with 3 mM H2O2. Thiol compounds and free radicals scavengers prevented the Fenton systems effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione, DL-dithiothreitol, cysteine and N-acetyl-L-cysteine entirely prevented the effect of the H2O2/Fe(II) system, mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, reduced glutathione, DL-dithiothreitol and N-acetyl-L-cysteine protected 100%, cysteine, histidine and mannitol protected 28, 34 and 48% respectively, whereas ethanol was ineffective. With the H2O2/Cu(II) system and T. cruzi topoisomerase, DL-dithiothreitol and histidine protected 100% and 60%, respectively but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by H2O2/Fe(II) or H2O2/Cu(II) systems was irreversible since they were not reverted by the more effective enzyme protectors. It is suggested that topoisomerases could act either as scavengers of "reactive oxygen species" (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species "in situ".

Acción inhibitoria de sistemas Fenton sobre la topoisomerasa I de Trypanosoma cruzi y Crithidia fasciculata / Effect of Fenton systems on Trypanosoma cruzi and Crithidia fasciculata topoisomerase I

Los sistemas Fenton (H2O2/Fe o H2O2/Cu) fueron capaces de inhibir la actividad topoisomerasa I de extractos crudos de Trypanosoma cruzi y Crithidia fasciculata. El agregado de compuestos de tioles o complejantes de metales, modificó la inhibición y dicho efecto dependió del metal y del origen de la enzima. El glutation reducido, DL-ditiotreitol, y N-aceti-L-cisteína 1 mM fueron efectivos protectores frente a la inhibición, inducida por el sistema H2O2/Fe, de la actividad presente en T.cruzi, el manitol protegió 37 por ciento, mientras que la histidina y etanol fueron inefectivos. Con la topoisomerasa de C.fasciculata, glutatión reducido, DL-ditiotreitol y N-acetil L-cisteína protegieron 100 por ciento a la enzima de la acción deletérea del sistema Fenton (Fe), los compuestos manitol, histidina y cisteína 1 mM protegieron 48, 34 y 28 por ciento, respectivamente, mientras que el etanol 4 mM fue inefectivo. Con el sistema H2O2/Cu y la enzima de T.cruzi, el DL-ditiotreitol y la histidina 1mM protegieron 100 y 60 por ciento, respectivamente, los otros protectores ensayados fueron menos efectivos. Resultados semejantes se obtuvieron con la topoisomerasa de C.fasciculata. La disminución por sistemas Fenton de la actividad topoisomerasa I de los extractos resultó no ser revertida por posterior incubación con los compuestos que tuvieron efecto protector. Se sugiere que la estructura molecular de la proteína podría actuar como secuestrante de radicales libres generados por los sismtemas Fenton o mediante la unión de metales como el Cu o Fe, facilitando la generación de los mismos in situ. Ambos mecanismos conducirían a la inactivación de la misma.

Acción inhibitoria de sistemas Fenton sobre la topoisomerasa I de Trypanosoma cruzi y Crithidia fasciculata / Effect of Fenton systems on Trypanosoma cruzi and Crithidia fasciculata topoisomerase I

Los sistemas Fenton (H2O2/Fe o H2O2/Cu) fueron capaces de inhibir la actividad topoisomerasa I de extractos crudos de Trypanosoma cruzi y Crithidia fasciculata. El agregado de compuestos de tioles o complejantes de metales, modificó la inhibición y dicho efecto dependió del metal y del origen de la enzima. El glutation reducido, DL-ditiotreitol, y N-aceti-L-cisteína 1 mM fueron efectivos protectores frente a la inhibición, inducida por el sistema H2O2/Fe, de la actividad presente en T.cruzi, el manitol protegió 37 por ciento, mientras que la histidina y etanol fueron inefectivos. Con la topoisomerasa de C.fasciculata, glutatión reducido, DL-ditiotreitol y N-acetil L-cisteína protegieron 100 por ciento a la enzima de la acción deletérea del sistema Fenton (Fe), los compuestos manitol, histidina y cisteína 1 mM protegieron 48, 34 y 28 por ciento, respectivamente, mientras que el etanol 4 mM fue inefectivo. Con el sistema H2O2/Cu y la enzima de T.cruzi, el DL-ditiotreitol y la histidina 1mM protegieron 100 y 60 por ciento, respectivamente, los otros protectores ensayados fueron menos efectivos. Resultados semejantes se obtuvieron con la topoisomerasa de C.fasciculata. La disminución por sistemas Fenton de la actividad topoisomerasa I de los extractos resultó no ser revertida por posterior incubación con los compuestos que tuvieron efecto protector. Se sugiere que la estructura molecular de la proteína podría actuar como secuestrante de radicales libres generados por los sismtemas Fenton o mediante la unión de metales como el Cu o Fe, facilitando la generación de los mismos in situ. Ambos mecanismos conducirían a la inactivación de la misma. (AU)

Acción inhibitoria de sistemas Fenton sobre la topoisomerasa I de Trypanosoma cruzi y Crithidia fasciculata. / [Inhibitory action of Fenton systems on topoisomerase I from Trypanosoma cruzi and Crithidia fasciculata]]

Fenton systems (H2O2/Fe(II) or H2O2/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71

inactivation was produced by 25 mM Fe(II) or Cu(II) with 3 mM H2O2. Thiol compounds and free radicals scavengers prevented the Fenton systems effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione, DL-dithiothreitol, cysteine and N-acetyl-L-cysteine entirely prevented the effect of the H2O2/Fe(II) system, mannitol protected 37

, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, reduced glutathione, DL-dithiothreitol and N-acetyl-L-cysteine protected 100

, cysteine, histidine and mannitol protected 28, 34 and 48

respectively, whereas ethanol was ineffective. With the H2O2/Cu(II) system and T. cruzi topoisomerase, DL-dithiothreitol and histidine protected 100

and 60

, respectively but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by H2O2/Fe(II) or H2O2/Cu(II) systems was irreversible since they were not reverted by the more effective enzyme protectors. It is suggested that topoisomerases could act either as scavengers of [quot ]reactive oxygen species[quot ] (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species [quot ]in situ[quot ].

[Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use]. / Citotoxicidad de la beta-lapachona: una o-naftoquinona con posibles usos terapéuticos.

beta-lapachone (beta-lap) is a lipophilic o-naphthoquinone isolated from the bark of the lapacho tree. Initial observations proved its capability for inhibiting growth of Yoshida tumor and Walker 256 carcinosarcoma. beta-Lap redox-cycling in the presence of reductants and oxygen yields "reactive oxygen species" (ROS: O2-, OH and H2O2) which cytotoxicity led to assume its role in beta-lap activity in cells. beta-Lap inhibited DNA synthesis in Trypanosoma cruzi as well as topoisomerases I and II, poly(ADP-ribose) polymerase (PARP) in different cells. These enzymes are essential for maintaining DNA structure. beta-Lap inhibited growth of a large variety of tumor cells including epidermoid laringeal cancer, prostate, colon, ovary and breast cancer and also different types of leukemia cells. Advances in knowledge of apoptosis ("programmed cell death") and necrosis provided useful information for understanding the mechanism of beta-lap cytotoxicity. Thiol-dependent proteases (Calpaine), kinases (e.g. c-JUN NH2-terminal kinase), caspases and nucleases are involved in beta-lap cytotoxicity. These enzymes activity, as well as ROS production by beta-lap redox-cycling, would be essential for beta-lap cytotoxicity. Diaphorase and NAD(P)H-quinone reductase, which catalyse beta-lap redox-cycling and ROS production, seem to play an essential role in beta-lap activity. On these grounds, clinical applications of beta-lap have been suggested.

Citotoxicidad de la beta-lapachona: una o-naftoquinona con posibles usos terapéuticos. / [Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use]

beta-lapachone (beta-lap) is a lipophilic o-naphthoquinone isolated from the bark of the lapacho tree. Initial observations proved its capability for inhibiting growth of Yoshida tumor and Walker 256 carcinosarcoma. beta-Lap redox-cycling in the presence of reductants and oxygen yields [quot ]reactive oxygen species[quot ] (ROS: O2-, OH and H2O2) which cytotoxicity led to assume its role in beta-lap activity in cells. beta-Lap inhibited DNA synthesis in Trypanosoma cruzi as well as topoisomerases I and II, poly(ADP-ribose) polymerase (PARP) in different cells. These enzymes are essential for maintaining DNA structure. beta-Lap inhibited growth of a large variety of tumor cells including epidermoid laringeal cancer, prostate, colon, ovary and breast cancer and also different types of leukemia cells. Advances in knowledge of apoptosis ([quot ]programmed cell death[quot ]) and necrosis provided useful information for understanding the mechanism of beta-lap cytotoxicity. Thiol-dependent proteases (Calpaine), kinases (e.g. c-JUN NH2-terminal kinase), caspases and nucleases are involved in beta-lap cytotoxicity. These enzymes activity, as well as ROS production by beta-lap redox-cycling, would be essential for beta-lap cytotoxicity. Diaphorase and NAD(P)H-quinone reductase, which catalyse beta-lap redox-cycling and ROS production, seem to play an essential role in beta-lap activity. On these grounds, clinical applications of beta-lap have been suggested.

Semiquinone production by lipophilic o-naphthoquinones.

Several ß-lapachone analogues, the o-naphthoquinones CG 10-248, CG 9-442 and CG 8-935, were reduced to their semiquinones by sodium borohydride, the liver NADPH-P450 reductase system and Crithidia fasciculata cells, in anaerobic solutions. ESR spectra of the radical anions showed hyperfine spin couplings located at protons of the naphthalene ring. Borohydride reduction of another o-naphthoquinone, mansonone E, yielded spin couplings located at the naphthalene and methyl groups protons. The symmetrical polarized carbonyl groups were essential for electron capture and semiquinone production. These observations support the idea that quinones are capable of redox-cycling and oxygen radical generation.

Redox cycling of o-naphthoquinones in trypanosomatids. Superoxide and hydrogen peroxide production.

beta-Lapachone and structurally related lipophilic o-naphthoquinones, namely, CG 8-935, CG 9-442, CG 10-248 and mansonones A, C, E, and F, were investigated for redox cycling, production of reactive oxygen species, and cytotoxicity in the trypanosomatids Crithidia fasciculata and Leptomonas seymouri. Structural analysis of the assayed quinones indicated that a tricyclic structure, including a naphthalene ring, a 1,2b or 1,8bc pyran ring, and two ortho-carbonyl groups were required for quinone activities. The contribution of oxygen radical production to quinone cytotoxicity was supported by: (a) spectroscopic observation of quinone redox cycling; (b) production of the semiquinone radical; (c) H2O2 and O2- production; (d) the effect of beta-lapachone on thiol pools in C. fasciculata; (e) the effect of quinones on cell respiration; (f) superoxide dismutase inactivation after incubation of C. fasciculata with CG 8-935; and (g) the effect of quinones on cell growth.

Effect of 5-nitroindole on adenylate energy charge, oxidative phosphorylation, and lipid peroxidation in rat hepatocytes.

5-Nitroindole (NI), a mutagenic nitroarene, was assayed for cytotoxic effects on rat hepatocytes. After incubation with 25-100 microM NI, the adenylate energy charge of the hepatocytes decreased significantly as a result of the decrease in ATP and the increase in AMP. ATP depletion correlated well with the effects of NI on mitochondrial electron transfer and energy transduction in hepatocytes. Thus, NI (a) inhibited the antimycin-sensitive hepatocyte respiration; (b) inhibited NADH oxidation by disrupted hepatocyte mitochondria; (c) inhibited L-malate-L-glutamate oxidation by ADP-supplemented mitochondria; (d) in the absence of ADP, stimulated the same substrates and also succinate oxidation by mitochondria; (e) released the latent ATPase activity of mitochondrial F1F0-ATP synthase; (f) shifted the redox level of reduced cytochromes (c + c1) and b towards the oxidized state; (g) inhibited NADH oxidation by disrupted mitochondria in the vicinity of the NADH-dehydrogenase flavoprotein; (h) inhibited Ca2+ uptake by mitochondria using L-malate-L-glutamate as an energy source; (i) inhibited valinomycin-induced, endogenously energized K+ uptake, with little effect on the ATP-induced uptake; and (j) inhibited the MgATP-dependent contraction of Ca(2+)-swollen mitochondria. NI inhibited lipid peroxidation in hepatocytes and also in substrate-supplemented liver microsomes and mitochondria, thus ruling out hydroperoxides as a cause of NI cytotoxicity. Long-term incubation with NI produced loss of hepatocyte viability, as indicated by lactate dehydrogenase leakage.

[Inhibition of oxidative phosphorylation in Crithidia fasciculata and Trypanosoma cruzi by lipophilic o-quinones and nifurtimox]. / Inhibición de la oxidación fosforilante en Crithidia fasciculata y Trypanosoma cruzi por o-quinonas lipofílicas y nifurtimox.

ATP and ADP levels were determined in Crithidia fasciculata and Trypanosoma cruzi. The nucleotide levels in crithidia or epimastigotes at the stationary phase of growth were, in nmol/10(8) cells, 15-40, and 3-7, for ATP and ADP, respectively. Incubation with the lipophilic o-naphthoquinones CG 8-935, CG 9-442 and CG 10-248 or the anti-chagasic nitrofuran nifurtimox, significantly decreased ATP level, with non-significant variations of the ADP level. The kinetics of ATP variation showed an initial 1-2 h lag and the diminution of the ATP level reached maximum value after 4-6 h incubation. Addition of L-glutamate or D-glucose as energy sources produced 2- or 3-fold increase of ATP level, after incubation the protozoa for 4-6 h with the corresponding substrates. Quinones and nifurtimox strongly depressed D-glucose or L-glutamate effects. Buthionine sulfoximine an inhibitor of glutathione biosynthesis, enhanced the effect of nifurtimox on ATP level in Crithidia fasciculata. It is concluded that by inhibiting ATP synthesis, either directly or-through oxygen radicals, the assayed drugs produced their cytotoxic action.

Inhibición de la oxidación fosforilante en Crithidia fasciculata y Trypanosoma cruzi por o-quinonas lipofílicas y nifurtimox / [Inhibition of oxidative phosphorylation in Crithidia fasciculata and Trypanosoma cruzi by lipophilic o-quinones and nifurtimox]

ATP and ADP levels were determined in Crithidia fasciculata and Trypanosoma cruzi. The nucleotide levels in crithidia or epimastigotes at the stationary phase of growth were, in nmol/10(8) cells, 15-40, and 3-7, for ATP and ADP, respectively. Incubation with the lipophilic o-naphthoquinones CG 8-935, CG 9-442 and CG 10-248 or the anti-chagasic nitrofuran nifurtimox, significantly decreased ATP level, with non-significant variations of the ADP level. The kinetics of ATP variation showed an initial 1-2 h lag and the diminution of the ATP level reached maximum value after 4-6 h incubation. Addition of L-glutamate or D-glucose as energy sources produced 2- or 3-fold increase of ATP level, after incubation the protozoa for 4-6 h with the corresponding substrates. Quinones and nifurtimox strongly depressed D-glucose or L-glutamate effects. Buthionine sulfoximine an inhibitor of glutathione biosynthesis, enhanced the effect of nifurtimox on ATP level in Crithidia fasciculata. It is concluded that by inhibiting ATP synthesis, either directly or-through oxygen radicals, the assayed drugs produced their cytotoxic action.

Inhibición de la oxidación fosforilante en Crithidia fasciculata y Trypanosoma cruzi por o-quinonas lipofílicas y nifurtimox. / [Inhibition of oxidative phosphorylation in Crithidia fasciculata and Trypanosoma cruzi by lipophilic o-quinones and nifurtimox]

ATP and ADP levels were determined in Crithidia fasciculata and Trypanosoma cruzi. The nucleotide levels in crithidia or epimastigotes at the stationary phase of growth were, in nmol/10(8) cells, 15-40, and 3-7, for ATP and ADP, respectively. Incubation with the lipophilic o-naphthoquinones CG 8-935, CG 9-442 and CG 10-248 or the anti-chagasic nitrofuran nifurtimox, significantly decreased ATP level, with non-significant variations of the ADP level. The kinetics of ATP variation showed an initial 1-2 h lag and the diminution of the ATP level reached maximum value after 4-6 h incubation. Addition of L-glutamate or D-glucose as energy sources produced 2- or 3-fold increase of ATP level, after incubation the protozoa for 4-6 h with the corresponding substrates. Quinones and nifurtimox strongly depressed D-glucose or L-glutamate effects. Buthionine sulfoximine an inhibitor of glutathione biosynthesis, enhanced the effect of nifurtimox on ATP level in Crithidia fasciculata. It is concluded that by inhibiting ATP synthesis, either directly or-through oxygen radicals, the assayed drugs produced their cytotoxic action.

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by nitrofuran compounds.

(5-Nitro-2-furfurylidene)amino compounds bearing triazol-4-yl, benzimidazol-1-yl, pyrazol-1-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-1-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-1-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-1-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-1-yl and substituted triazol-4-yl derivatives.

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by beta-lapachone and related naphthoquinones.

The lipophilic o-naphthoquinones beta-lapachone, 3,4-dihydro-2-methyl-2-ethyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 8-935), 3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 9-442), and 3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione (CG 10-248) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P-450 destruction; (c) inhibited microsomal aniline 4-hydroxylase, aminopyrine N-demethylase and 7-ethoxycoumarin deethylase; (d) did not inhibit the ascorbate- and tert-butyl hydroperoxide-dependent lipid peroxidation and the cumenyl hydroperoxide-linked aniline 4-hydroxylase reaction; and (e) stimulated NADPH oxidation, superoxide anion radical generation and Fe(III)ADP reduction by NADPH-supplemented microsomes. In the presence of ascorbate, the same o-naphthoquinones stimulated oxygen uptake and semiquinone formation, as detected by ESR measurements. The p-naphthoquinones alpha-lapachone and menadione were relatively less effective than the o-naphthoquinones. These observations support the hypothesis that, in the micromolar concentration range, o-naphthoquinones inhibit microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions, by diverting reducing equivalents from NADPH to dioxygen.

Generation of radical anions of nifurtimox and related nitrofuran compounds by ascorbate.

Nifurtimox analogues bearing triazol-4-yl, benzimidazol-1-yl, triazin-4-yl or related groups as counterpart of the (5-nitro-2-furfurylidene) amino group were reduced to their nitro anion radicals by ascorbate in anaerobic solutions at high pH. The ESR spectra of the radical anions showed hyperfine spin couplings restricted to the nitrofuran moiety. With these compounds, the spin density at the nitro group was greater than with nifurtimox, nitrofurazone and nitrofurantoin. At neutral pH, solutions containing ascorbate and nitrofuran derivatives consumed oxygen, the compounds bearing unsaturated nitrogen heterocycles being the most effective. Superoxide dismutase and catalase decreased the rate of oxygen consumption, thus demonstrating the production of superoxide and hydrogen peroxide, respectively. NMR spectra of the triazol-4-yl and triazin-4-yl nitrofuran derivatives showed a deshielding effect for the azomethine proton, which was undetectable with nifurtimox and nitrofurazone.

Nitrofuran inhibition of microsomal lipid peroxidation.

Two nitrofuran compounds, nifurtimox and nitrofurantoin, inhibited in a concentration-dependent manner the NADPH-, iron-induced lipid peroxidation in rat liver microsomes, as shown by the decreased rate of MDA accumulation. Other nitro compounds (benznidazole and chloramphenicol) were relatively inactive. Nifurtimox inhibition affected polyenoic fatty acids and cytochrome P-450 degradation that follows lipid peroxidation. The ascorbate- or tert-butyl hydroperoxide-dependent lipid peroxidations were much less inhibited than the NADPH-dependent one. Nifurtimox and nitrofurantoin, but not benznidazole and chloramphenicol, strongly stimulated the microsomal NADPH-oxidase activity, thus supporting electron diversion, as the main cause of the inhibition of peroxidation initiation.