Myeloperoxidase-generated phenothiazine cation radicals inactivate Trypanosoma cruzi dihydrolipoamide dehydrogenase

Peroxidase/H2O2/phenothiazine systems irreversibly inhibit Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH). Inactivation of the parasite enzyme depended on (a) phenothiazine structure; (b) peroxidase nature; (c) incubation time and (d) the presence of a cation radical scavenger. With the myeloperoxidase/H2O2/system, promazine, trimeprazine, thioridazine, promethiazine, prochlorperazine, chlorpromazine and perphenazine were the most effective derivatives out of twelve phenothiazines studied. An electronegative substituent at position 2 of the phenothiazine ring such as Cl, or trifluoromethyl, propionyl and nitrile groups decreased or nullified phenothiazine activity. Myeloperoxidase/H2O2/, horseradish peroxidase/H2O2/, and myoglobin/H2O2/systems activated phenothiazines producing the corresponding cation radicals, myeloperoxidase being the most selective one with respect to phenothiazine structure. The myoglobin/H2O2/system activated phenothiazines that were scarcely active or inactivate with the MPO/H2O2/system, such as the trifluoromethyl derivatives. Production of phenothiazine cation radicals was demonstrated by optical spectroscopy. Phenothiazine cation radical stability depended on their structure as illustrated by promazine and thioridazine. Thiol compounds (GSH, N-acetyl-cysteine and penicillamine), aromatic aminoacids (L-tyrosine, L-tryptophan, and the corresponding peptides) and ascorbate scavenged phenothiazine cation radicals, thus preventing LADH inactivation. Comparison of the summarized phenothiazine effects with those of phenothiazines on T. cruzi suggest the role of cation radicals in phenothiazines chemotherapeutic actions.

Inactivation of Trypanosoma cruzi dihydrolipoamide dehydrogenase by leukocyte myeloperoxidase systems: role of hypochloride and nitrite related radicals

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.

Inactivacion de la dihidrolipoamida deshidrogenasa de miocardio por Cu(II) y peroxido de hidrogeno / Inactivation of myocardial dihydrolipoamide dehydrogenase by Cu(II) and hydrogen peroxide

Se estudió la inactivación de la dihidrolipoamida deshidrogenasa de corazón porcino (LipDH) por oxi-radicales, generados por Cu(II) suplementado o no con H2O2 (sistema SF-CU(II) o ácido ascórbico (sistema Cu(II)-Asc). As concentraciones utilizadas fueron: 2,5-10 µM Cu(II): 3,0 mM H2O2 y 0,5 mM ascorbato. Después de 5 minutos de incubación, la actividad lipoamida reductasa de la LipDH decayó de manera irrevedrsible: 83-98 por ciento con SF-Cu(II) y Cu(II)-Asc o 46-53 por ciento con Cu(II). La actividad diaforasa aumentó, demostrando un daño localizado de los grupos SH de la LipDH. NAD+, dihidrolipoamida, GSSG, CAPTOPRIL, complejantes de CU(II) (DL-histidina, batocuproína, EDTA y DETAPAG), la tripanotiona y el alopurinol, protegieron la LipDH frente al SF-Cu(II). El GSH, el ditiotretol, la N-acetilcisteína, la mercaptopropionilglicina, la DL-penicilamina y la L-cisteína, protegieron LipDH frente al CU(II) solamente. NADH, ADP (no ATP), OH-DOPAMINA, DOPA, DOPAC y catecol, aumentaron la inactivación de LipDH por el SF-Cu(II) mientras que OH-DOPAMINA aumentó el efecto del Cu(II). Se discute la acción de los oxi-radicales generados por Cu(II), como causa del daño miocárdico por la reoxigenación post-isquemia