title: Phenothiazine radicals inactivate Trypanosoma cruzi dihydrolipoamide dehydrogenase: enzyme protection by radical scavengers.
abstract: Phenothiazine cation radicals (PTZ+*) irreversibly inactivated Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH). These radicals were obtained by phenothiazine (PTZ) peroxidation with myeloperoxidase (MPO) or horseradish peroxidase (HRP/H2O2) systems. LADH inactivation depended on PTZ structure and incubation time. After 10 min incubation of LADH with the MPO-dependent systems, promazine, trimeprazine and thioridazine were the most effective; after 30 min incubation, chlorpromazine, prochlorperazine and promethazine were similarly effective. HRP-dependent systems were equally or more effective than the corresponding MPO-dependent ones. Chloro, trifluoro, propionyl and nitrile groups at position 2 of the PTZ ring significantly decreased molecular activity, specially with the MPO/H2O2 systems. Comparison of inactivation values for LADH and T. cruzi trypanothione reductase demonstrated a greater sensitivity of LADH to chlorpromazine and perphenazine and a 10-fold lower sensitivity to promazine, thioridazine and trimeprazine. Alkylamino, alkyl-piperidinyl or alkyl-piperazinyl groups at position 10 modulated PTZ activity to a limited degree. Production of PTZ+* radicals was demonstrated by optical and ESR spectroscopy methods. PTZ+* radicals stability depended on their structure as demonstrated by promazine and thioridazine radicals. Thiol compounds such as GSH and N-acetylcysteine, L-tyrosine, L-tryptophan, the corresponding peptides, ascorbate and Trolox, prevented LADH inactivation by the MPO/H2O2/thioridazine system, in close agreement with their action as PTZ+* scavengers. NADH (not NAD+) produced transient protection of LADH against thioridazine and promazine radicals, the protection kinetics being affected by the relatively fast rate of NADH oxidation by these radicals. The role of the observed effects of PTZ radicals for PTZ cytotoxicity is discussed.
authors.affiliation: Bioenergetics Research Centre, School of Medicine, University of Buenos Aires, Paraguay 2155, 1121-Bueno Aires, Argentina.
authors.forename: José
authors.initials: J
authors.lastname: Gutiérrez-Correa
authors.forename: R Luise
authors.initials: RL
authors.lastname: Krauth-Siegel
authors.forename: Andrés O M
authors.initials: AO
authors.lastname: Stoppani
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology; D014150:Antipsychotic Agents / Q000494:pharmacology; D002412:Cations; D002746:Chlorpromazine / Q000494:pharmacology; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016166:Free Radical Scavengers / Q000378:metabolism / Q000494:pharmacology*; D005609:Free Radicals; D005978:Glutathione / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D010546:Perphenazine / Q000494:pharmacology; D010640:Phenothiazines / Q000737:chemistry / Q000494:pharmacology*; D011725:Pyridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D000975:Antioxidants; D014150:Antipsychotic Agents; D002412:Cations; D016166:Free Radical Scavengers; D005609:Free Radicals; D010640:Phenothiazines; D011725:Pyridines; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D009243:NAD; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D009195:Peroxidase; D008058:Dihydrolipoamide Dehydrogenase; D010546:Perphenazine; D005978:Glutathione; C031637:phenothiazine; D010100:Oxygen; D002746:Chlorpromazine