Reduction of nitrofuran compounds by heart lipoamide dehydrogenase: role of flavin and the reactive disulfide groups.

In order to elucidate the mechanism of the biological activation of nitrofurans, the interaction of these compounds with lipoamide dehydrogenase (LipDH)** was investigated. LipDH catalysed one-electron reduction of several nitrofuran derivatives. The reaction could be demonstrated spectroscopically and was enhanced by cadmium, arsenite and anaerobiosis. The role of flavin in the nitroreductase activity was supported by (a) the nitrofuran effect on the spectral properties of anaerobic, arsenite-inhibited, NADH-reduced LipDH; (b) FAD catalytic activity in a NADH-nitrofuran model system; and (c) the nitroreductase activity of LipDH monomer. Two-electron nitrofuran reduction to less oxidized products was inhibited by cadmium, arsenite and NAD+. The possible role of reactive nitrosofuran derivatives as intermediates of the nitrofuran reduction sequence was supported by the LipDH capability for catalysing 2-nitroso-1-naphthol redox-cycling. The nitroso naphthol reduction was inhibited by cadmium and arsenite, like the two-electron nitrofuran reduction.

Superoxide anion production by lipoamide dehydrogenase redox-cycling: effect of enzyme modifiers.

Redox-cycling of porcine heart lipoamide dehydrogenase in the presence of NADH and oxygen produced O2-. (NADH-oxidase activity) as demonstrated by (a) reduction of cytochrome c; (b) reduction of the Fe(III)-ADP complex; (c) lucigenin luminescence and (d) the inhibitory effect of superoxide dismutase. NAD+ and p-chloromercuribenzoate inhibited O2-. generation whereas arsenite enhanced it. Comparison of heart and yeast enzyme preparations revealed a close correlation between lipoamide reductase and NADH-oxidase activities. It is concluded that O2-. production is a molecular property of lipoamide dehydrogenase.

Catalysis of nitrofuran redox-cycling and superoxide anion production by heart lipoamide dehydrogenase.

Heart lipoamide dehydrogenase (LADH) catalyzed redox-cycling and O2-. production by (5-nitro-2-furfurylidene)amino derivatives using NADH as electron donor. NADH was a much more effective electron donor than NADPH for the nitroreductase activity. O2-. production was demonstrated by cytochrome c reduction, adrenochrome formation and the effect of superoxide dismutase. Under optimum conditions, nitroreductase activity was about 1% of LADH activity. One electron oxygen reduction and NADH oxidation correlated in 2:1 stoichiometry. The nitroreductase kinetics was in accordance with an ordered bi-bi mechanism. Nitrofuran derivatives bearing unsaturated five- or six-membered nitrogen heterocycles were more effective substrates than those bearing other groups, namely nifurtimox, nitrofurazone, nitrofurantoin and 5-nitro-2-furoic acid. Other nitro compounds (chloramphenicol, benznidazole, 2-nitroimidazole and 5-nitroindole) were ineffective. With the triazole, traizine and imidazole nitrofuran derivatives, the nitroreductase pH curve showed a maximum at pH 8.8, different from the pH optimum for the lipoamide reductase and diaphorase activities. Spectroscopic observations demonstrated pH-dependent structural changes in the triazole(I) and triazine derivatives which would affect their behavior as nitroreductase substrates. The nitroreductase activity was inhibited by p-chloromercuribenzoate and enhanced by cadmium and arsenite, whereas the NADH-induced LADH inactivation failed to affect the nitroreductase activity. In the absence of oxygen. LADH catalyzed nitrofuran reduction to products more reduced than the nitroanion, which were not reoxidized by oxygen. The anaerobic nitrofuran reduction was inhibited by cadmium and arsenite. The assayed nitrofuran compounds did not inhibit LADH lipoamide reductase activity, at variance with their action on glutathione reductase (Grinblat et al., Biochem Pharmacol 38: 767-772, 1989).

Nitrofuran inhibition of yeast and rat tissue glutathione reductases. Structure-activity relationships.

Nitrofuran derivatives bearing unsaturated five- or six-membered nitrogen heterocycles or related substituents were more effective inhibitors of yeast and rat tissue glutathione reductases than those bearing other groups, such as nifurtimox, nitrofurazone and 5-nitro-2-furoic acid. The inhibitory action proved independent of electron withdrawal from the reduced enzyme, as a consequence of redoxcycling of the nitro group. Uncompetitive kinetics was obtained with nitrofurantoin and nifurtimox. Most of the assayed nitrofurans inhibited the yeast enzyme Coenzyme A glutathione disulfide reductase activity, though less than oxidized glutathione reduction. The transhydrogenase activity was not inhibited to a significant degree. Benznidazole (a 2-nitroimidazole derivative), 2-nitroimidazole, 5-nitroindole and chloramphenicol did not inhibit glutathione reductase. Under the same experimental conditions, liver glutathione peroxidase was not affected by the nitro compounds.

[Diabetes and calcium transportation in liver mitochondria]. / Diabetes y transporte de calcio en mitocondrias hepáticas.

It is a well known fact that isolated, energized mitochondria take up large amounts of Ca2+, thus regulating their own internal Ca2+ concentration and modulating the activity of matrix dehydrogenases involved in the aerobic steps of glucose oxidation. The information available on biochemical alterations in diabetes is extensive but no data on Ca2+ transport alterations have been reported. Therefore, it seemed of interest to study Ca2+ uptake and release (efflux) by liver mitochondria of diabetic rats, in relation to other metabolic parameters representing the energization state of the inner mitochondrial membrane. Rats (male; 200 +/- 20 g body weight) were injected with streptozotocin (65 mg/kg) and after 1-3 months, liver mitochondria were isolated and suspended in an isotonic medium supplemented with 3.0 microM rotenon, 5.0 mM succinate (the energy source), 50 microM Antipyrylazo III and CaCl2 (20-100 microM Ca2+). Ca2+ uptake was monitored by the decrease of the Ca2(+)-Antipyrylazo III complex concentration, measured spectrophotometrically at 720-790 nm and 30 degrees C. The initial rates of Ca2+ uptake (in nmol Ca2+/min/mg of protein; average +/- S.E., n = 5) were as follows (in parenthesis, initial [Ca2+] microM): normal mitochondria, 171 +/- 20 (20); 207 +/- 13 (40); 233 +/- 22 (60) and 237 +/- 14 (100); diabetic mitochondria, 114 +/- 13; 134 +/- 22; 186 +/- 7 and 184 +/- 14, respectively. Accordingly, the decrease of Ca2+ uptake activity was 33, 36, 20 and 22 (%), respectively (P less than 0.05 at all [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetes y transporte de calcio en mitocondrias hepáticas. / [Diabetes and calcium transportation in liver mitochondria]

It is a well known fact that isolated, energized mitochondria take up large amounts of Ca2+, thus regulating their own internal Ca2+ concentration and modulating the activity of matrix dehydrogenases involved in the aerobic steps of glucose oxidation. The information available on biochemical alterations in diabetes is extensive but no data on Ca2+ transport alterations have been reported. Therefore, it seemed of interest to study Ca2+ uptake and release (efflux) by liver mitochondria of diabetic rats, in relation to other metabolic parameters representing the energization state of the inner mitochondrial membrane. Rats (male; 200 +/- 20 g body weight) were injected with streptozotocin (65 mg/kg) and after 1-3 months, liver mitochondria were isolated and suspended in an isotonic medium supplemented with 3.0 microM rotenon, 5.0 mM succinate (the energy source), 50 microM Antipyrylazo III and CaCl2 (20-100 microM Ca2+). Ca2+ uptake was monitored by the decrease of the Ca2(+)-Antipyrylazo III complex concentration, measured spectrophotometrically at 720-790 nm and 30 degrees C. The initial rates of Ca2+ uptake (in nmol Ca2+/min/mg of protein; average +/- S.E., n = 5) were as follows (in parenthesis, initial [Ca2+] microM): normal mitochondria, 171 +/- 20 (20); 207 +/- 13 (40); 233 +/- 22 (60) and 237 +/- 14 (100); diabetic mitochondria, 114 +/- 13; 134 +/- 22; 186 +/- 7 and 184 +/- 14, respectively. Accordingly, the decrease of Ca2+ uptake activity was 33, 36, 20 and 22 (

), respectively (P less than 0.05 at all [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium transport and energy coupling in diabetic rat liver mitochondria.

Liver mitochondria from chronic diabetic rats took up Ca ions at a significantly slower rate than their normal counterparts. The same mitochondria, inhibited with ruthenium red, released Ca ions at a faster rate than normal mitochondria. In good agreement with the decrease of Ca ions uptake, measurement of the inner membrane potential by the safranine method yielded a significantly lower value with diabetic than with normal mitochondria. Respiratory control and P/O ratios were also decreased in diabetic mitochondria.

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.

Decreased rate of ketone-body oxidation and decreased activity of D-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats.

Heart mitochondria from chronically diabetic rats ('diabetic mitochondria'), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats ('normal mitochondria'). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.

Disminucion de los citocromos y la biosintesis proteica mitocondrial en musculo esqueletico de ratas diabeticas. / Decrease cytochromes and mitochondrial protein biosynthesis in skeletal muscle of diabetic rats

Se determino el contenido en citocromos aa3, b, c y c1 en mitocondrias de musculo esqueletico, higado y corazon de ratas diabeticas por inyeccion de estreptozotocina (65 mg/kg, dosis unica, intravenosa), 30 dias despues de la iniciacion de la diabetes. Se aislaron las mitocondrias y se determino su contenido en citocromos. Solamente en las mitocondrias de musculo esqueletico se comprobo disminucion significativa (p < 0.001) de los citocromos, especialmente aa3 y b (en las mitocondrias de higado hubo aumento de los citocromos aa3).La variacion de los citocromos musculares concordo con la disminucion de la sintesis de proteinas, comprobada utilizando el procedimiento de Favalukes y col. para medir la incorporacion de aminoacidos en proteinas mitocondriales

Disminucion de los citocromos y la biosintesis proteica mitocondrial en musculo esqueletico de ratas diabeticas. / Decrease cytochromes and mitochondrial protein biosynthesis in skeletal muscle of diabetic rats

Se determino el contenido en citocromos aa3, b, c y c1 en mitocondrias de musculo esqueletico, higado y corazon de ratas diabeticas por inyeccion de estreptozotocina (65 mg/kg, dosis unica, intravenosa), 30 dias despues de la iniciacion de la diabetes. Se aislaron las mitocondrias y se determino su contenido en citocromos. Solamente en las mitocondrias de musculo esqueletico se comprobo disminucion significativa (p < 0.001) de los citocromos, especialmente aa3 y b (en las mitocondrias de higado hubo aumento de los citocromos aa3).La variacion de los citocromos musculares concordo con la disminucion de la sintesis de proteinas, comprobada utilizando el procedimiento de Favalukes y col. para medir la incorporacion de aminoacidos en proteinas mitocondriales