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.

Inactivación de la lipoamida deshidrogenasa de miocardio por catecolaminas. Protección por captopril y otros tioles. / [Inactivation of the myocardial lipoamide dehydrogenase by catecholamines. Prevention by captopril and other thiol compounds]

Inactivation of lipoamide dehydrogenase (LipDH) by the Cu(II)/H2O2 Fenton system (SF-Cu(II): (5.0 microM Cu(II), 3.0 mM H2O2) was enhanced by catecholamines (CAs), namely, epinephrine, levoDOPA (DOPA), DOPAMINE, 6-hydroxyDOPAMINE (OH-DOPAMINE) and related compounds (DOPAC, CATECHOL, etc.). After 5 min incubation with the Cu(II)/H2O2/CA system (0.4 mM CA), the enzyme activity decayed as indicated by the following percentage values (mean +/- S.D.; in parenthesis, number of determinations): SF-Cu(II) alone, 43 +/- 10 (18); SF-Cu(II) + epinephrine, 80 +/- 9 (5); SF-Cu(II) + DOPA, 78 +/- 2 (4); SF + Cu(II) + DOPAMINE, 88 +/- 7 (5); SF-Cu(II) + OH-DOPAMINE 87 +/- 6 (7); SF-Cu(II) +/- DOPAC, 88 +/- 3 (6); SF-Cu(II) + catechol, 85 +/- 6 (5). In all cases P < 0.05, with respect to the SF-Cu(II) control sample. CAs effect was concentration-dependent and at the 0-100 microM concentration range, it varied with the CA structure. Above the 100 microM concentration, CAs were equally effective and produced 90-100

enzyme, inactivation (Figure 2). In the absence of oxy-radical generation, the enzyme specific activity (mean +/- S.D.) was 149 +/- 10 (24) mumol NADH/min/mg protein. Assay of HO. production by the Cu(II)/H2O2/CA system in the presence of deoxyribose (TBA assay) yielded values much greater than those obtained omitting CA. Hydroxyl radical production depended on the presence of Cu(II) and H2O2 and significant H. values were obtained with OH-DOPAMINE, DOPAC, epinephrine, DOPAMINE, DOPA and catecol supplemented systems (Table 2). LipDH (1.0 microM) inhibited 50-80

deoxyribose oxidation, the inhibition depending on the CA structure (Table 2). Native catalase (20 micrograms/ml) and bovine serum albumin (40 micrograms/ml) effectively prevented LipDH inactivation by the Cu(II)/H2O2/CA system; denaturated catalase, SOD, 0.3 M mannitol, 6.0 mM ethanol and 0.2 M benzoate were less effective or did not protect LipDH (Table 3). Incubation of CAs with the Cu(II)/H2O2 system produced a time and Cu(II)-dependent destruction of CAs, the corresponding o-quinone, production as illustrated with epinephrine (figures 6 and 7), as illustrated with epinephrine and DOPAMINE (Table 4). These results support LipDH inactivation by (a) reduction of Cu(II) to Cu(I) by CAs followed by Cu-catalyzed production of HO. from H2O2; (b) CA oxidation followed by the corresponding o-quinone interaction with LipDH. CAPTOPRIL, N-acetylcysteine, mercaptopropionylglycine and penicillamine prevented to various degree LipDH inactivation by the Cu(II)/H2O2/CA systems (Table 1). The former was the most effective and 0.4 mM CAPTOPRIL prevented about 95-100

the effect of Cu(II)/H2O2/CA systems supplemented with epinephrine, DOPAMINE and OH-DOPAMINE (Figures 3 and Table 1). LipDH increased and CAPTOPRIL inhibited epinephrine oxidation by Cu(II)/H2O2 (Figures 4 and 5). Since un-physiological concentrations of CAs and Cu(II) may be released in the myocardium after ischemia-reperfusion, the summarized observations may contribute to explain myocardial damage in that condition.

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.

Effect of dyskinetoplastic agents on ultrastructure and oxidative phosphorylation in Crithidia fasciculata.

Ethidium bromide (EB) is an intercalating agent which binds specifically to the kinetoplast (mitochondrial) DNA (kDNA) of trypanosomatids. Accordingly, EB inhibits DNA replication, thus inducing dyskinetoplasty. Since in eukariotic organisms mitochondrial DNA encodes the genetic information for cytochromes b, aa3 and F0F1 ATPase, it seemed of interest to establish whether a similar effect occurs in Crithidia fasciculata, a trypanosomatid used for assay of potential trypanocidal drugs. Culturing of C. fasciculata in the presence of EB inhibited growth and induced dyskinetoplasty, as confirmed by electron microscopy. The kinetoplast of EB-cultured crithidia lost its characteristic arc shape, it was misplaced in the cell cytoplasm its matrix structure and membrane differentiation were specifically modified. Dyskinetoplasty decreased crithidia respiration and oxidative phosphorylation, as indicated by the lower ATP level, ATP/ADP ratio and adenylate energy charge. The interference of EB with kinetoplastic constituents synthesis was confirmed by the lack of action of EB on crithidia in the stationary phase of growth, that ruled out direct inhibition of oxidative phosphorylation enzymes. The lipophilic o-naphthoquinone beta-lapachone produced structural alterations in kinetoplast membranes, that correlated with inhibition of oxidative phosphorylation. These latter effects involved free radicals since they were prevented by free radical scavengers.