ABSTRACT
The review presents: a) a brief description of the disease; b) a summary of the most important metabolic targets so far identified in Trypanosome cruzi (T. cruzi) along with corresponding inhibitor compounds; c) the current state of knowledge on the trypanothione reductase system of trypanosomatids with reference to oxidative stress defenses; d) detailed discussions on T. cruzi trypanothione reductase inhibitors such as nitrofuranes, naphthoquinones and phenothiazines. As yet, the chemotherapy of Chagas' disease remains an unsolved problem. Further search for new drugs must continue by means of nucleating existing chemotherapy efforts.
Subject(s)
Chagas Disease/drug therapy , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Trypanocidal Agents/therapeutic use , Trypanosoma cruzi/drug effects , Trypanosomatina/drug effects , Animals , Humans , NADH, NADPH Oxidoreductases/metabolism , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/enzymology , Trypanosomatina/enzymologyABSTRACT
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".
Subject(s)
Crithidia fasciculata/enzymology , Enzyme Inhibitors/pharmacology , Hydrogen Peroxide/pharmacology , Iron/pharmacology , Protozoan Proteins/antagonists & inhibitors , Topoisomerase I Inhibitors , Trypanosoma cruzi/enzymology , Animals , Chelating Agents/pharmacology , Crithidia fasciculata/drug effects , Hydrogen Peroxide/antagonists & inhibitors , Iron/antagonists & inhibitors , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Species Specificity , Sulfhydryl Reagents/pharmacology , Trypanosoma cruzi/drug effectsABSTRACT
CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species. (AU)
Subject(s)
Humans , Male , RESEARCH SUPPORT, NON-U.S. GOVT , Apoptosis/drug effects , Hepatocytes/drug effects , Naphthoquinones/pharmacology , Naphthoquinones/toxicity , Apoptosis/physiology , Cell Surface Extensions/drug effects , Cell Surface Extensions/pathology , Cell Surface Extensions/ultrastructure , Cells, Cultured , Chromatin/drug effects , Chromatin/pathology , DNA Fragmentation/drug effects , DNA Fragmentation/physiology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum/ultrastructure , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Hydrogen Peroxide/metabolism , Intracellular Membranes/drug effects , Intracellular Membranes/pathology , Intracellular Membranes/ultrastructure , Microscopy, Electron , Microvilli/drug effects , Microvilli/pathology , Microvilli/ultrastructure , Mitochondria/drug effects , Mitochondria/pathology , Mitochondria/ultrastructure , Rats , Rats, WistarABSTRACT
CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species.
Subject(s)
Humans , Male , Apoptosis/drug effects , Hepatocytes/drug effects , Naphthoquinones/pharmacology , Naphthoquinones/toxicity , Apoptosis/physiology , Cells, Cultured , Chromatin/drug effects , Chromatin/pathology , Cell Surface Extensions/drug effects , Cell Surface Extensions/pathology , Cell Surface Extensions/ultrastructure , DNA Fragmentation/drug effects , DNA Fragmentation/physiology , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Microscopy, Electron , Intracellular Membranes/drug effects , Intracellular Membranes/pathology , Intracellular Membranes/ultrastructure , Microvilli/drug effects , Microvilli/pathology , Microvilli/ultrastructure , Mitochondria/drug effects , Mitochondria/pathology , Mitochondria/ultrastructure , Hydrogen Peroxide/metabolism , Rats , Rats, Wistar , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum/ultrastructureABSTRACT
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)
Subject(s)
Trypanosoma cruzi , Crithidia fasciculata , ArgentinaABSTRACT
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.
Subject(s)
Crithidia fasciculata , DNA Topoisomerases, Type I , Trypanosoma cruzi , ArgentinaABSTRACT
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 ].
ABSTRACT
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.
Subject(s)
Cations/pharmacology , Dihydrolipoamide Dehydrogenase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Peroxidase/pharmacology , Phenothiazines/chemistry , Protozoan Proteins/antagonists & inhibitors , Trypanocidal Agents/chemistry , Trypanosoma cruzi/enzymology , Amino Acids, Aromatic/pharmacology , Animals , Ascorbic Acid/pharmacology , Free Radical Scavengers/pharmacology , Free Radicals , Humans , Hydrogen Peroxide/metabolism , Peroxidase/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Structure-Activity Relationship , Sulfhydryl Compounds/pharmacology , Trypanosoma cruzi/drug effectsABSTRACT
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.(AU)
Subject(s)
Animals , Comparative Study , Humans , RESEARCH SUPPORT, NON-U.S. GOVT , Cations/pharmacology , Enzyme Inhibitors/pharmacology , Dihydrolipoamide Dehydrogenase/antagonists & inhibitors , Peroxidase/pharmacology , Phenothiazines/chemistry , Protozoan Proteins/antagonists & inhibitors , Trypanocidal Agents/chemistry , Trypanosoma cruzi/enzymology , Amino Acids, Aromatic/pharmacology , Ascorbic Acid/pharmacology , Free Radical Scavengers/pharmacology , Free Radicals , Hydrogen Peroxide/metabolism , Peroxidase/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Structure-Activity Relationship , Sulfhydryl Compounds/pharmacology , Trypanosoma cruzi/drug effectsABSTRACT
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.
Subject(s)
Animals , Humans , Cations , Dihydrolipoamide Dehydrogenase , Enzyme Inhibitors/pharmacology , Peroxidase , Phenothiazines , Protozoan Proteins/antagonists & inhibitors , Trypanocidal Agents , Trypanosoma cruzi , Ascorbic Acid/pharmacology , Amino Acids, Aromatic/pharmacology , Free Radical Scavengers , Free Radicals , Peroxidase , Hydrogen Peroxide/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Structure-Activity Relationship , Sulfhydryl Compounds , Trypanosoma cruziABSTRACT
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.
ABSTRACT
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.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Apoptosis/drug effects , Naphthoquinones/pharmacology , Neoplasms/drug therapy , Poly(ADP-ribose) Polymerases/metabolism , Reactive Oxygen Species/physiology , Animals , Antibiotics, Antineoplastic/therapeutic use , Carcinoma 256, Walker/drug therapy , Carcinoma 256, Walker/enzymology , Humans , Naphthoquinones/therapeutic use , Neoplasms/enzymology , Sarcoma, Yoshida/drug therapy , Sarcoma, Yoshida/enzymology , Topoisomerase I InhibitorsABSTRACT
Peroxidase/H2O2/phenothiazine systems produced irreversible inhibition (inactivation) of Trypanosoma cruzi trypanothione reductase (TR). The enzyme inactivation depended on (a) the incubation time of TR with the peroxidase/H2O2/phenothiazine system; (b) the peroxidase nature and (c) the phenothiazine structure. With the more effective peroxidase/H2O2/phenothiazine systems, TR inactivation kinetics presented a relatively fast initial phase, lasting for about 10 min, in which most of the enzyme activity disappeared. This phase was followed by a slower one and, after 30 min incubation, TR was totally inactivated. Three peroxidases were assayed as catalysts of TR inactivation: the horseradish peroxidase (HRP), leukocyte myeloperoxidase (MPO) and modified myoglobin (Mb). Under comparable experimental conditions, the peroxidase system activity decreased in the given order. With HRP systems, 10 microM Thioridazine (TRDZ), Promazine (PZ), Trimeprazine (TMPZ), Prochlorperazine (PCZ), Propionylpromazine (PPZ), Chlorpromazine (CPZ) and Perphenazine (PFZ), produced 95-100% inactivation of TR. With the MPO/H2O2 systems, PZ. TRDZ and TMPZ were the most effective. Under similar experimental condition, the Mb/H2O2/PZ,/TMPZ, /TRDZ and CPZ systems effectively inactivated TR. The presence of alkylamino, piperazinyl, or piperidinyl groups in PTZ N atom (position 10) and -Cl, -CF3, -SCH3, COCH2CH3 and -CN in position C2 exerted significant influence on phenothiazine activity. Glutathione (GSH) prevented TR inactivation by the HRP/H2O2/PZ and MPO/H2O2/PZ systems. The HRP/H2O2 and MPO/H2O2/phenothiazines systems generated the corresponding cationic radicals (FTZ.+) the stability of which was limited by their conversion into phenothiazine-sulfoxides (PTZ-SO). The latter ones were inactive on TR. GSH rapidly reacted with PTZ+.; thus producing cation radical detoxication. These reactions fit in well with GSH protection of TR against the peroxidase/H2O2/phenothiazine system, as well as with the FTZ.+ role in phenothiazine cytotoxicity.
Subject(s)
Antiprotozoal Agents/pharmacology , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Phenothiazines/pharmacology , Protozoan Proteins/antagonists & inhibitors , Trypanosoma cruzi/enzymology , Animals , Antiprotozoal Agents/chemistry , Cations/pharmacology , Free Radicals , Glutathione/metabolism , Hydrogen Peroxide/pharmacology , Kinetics , Molecular Structure , Oxidation-Reduction , Peroxidases/pharmacology , Phenothiazines/chemistry , Recombinant Fusion Proteins/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Crithidia fasciculata/drug effects , DNA, Kinetoplast/metabolism , Ethidium/pharmacology , Mitochondria/drug effects , Oxidative Phosphorylation/drug effects , Trypanocidal Agents/pharmacology , Adenosine Triphosphate/metabolism , Animals , Ca(2+) Mg(2+)-ATPase/metabolism , Crithidia fasciculata/physiology , Crithidia fasciculata/ultrastructure , DNA, Kinetoplast/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/ultrastructure , Naphthoquinones/pharmacology , Sulfhydryl Compounds/pharmacologyABSTRACT
Trypanosoma cruzi trypanothione reductase (TR) was irreversibly inhibited by peroxidase/H2O2 /phenothiazine (PTZ) systems. TR inactivation depended on (a) time of incubation with the phenothiazine system; (b) the peroxidase nature and (c) the PTZ structure and concentration. With the most effective systems, TR inactivation kinetics were biphasic, with a relatively fast initial phase during which about 75% of the enzyme activity was lost, followed by a slower phase leading to total enzyme inactivation. GSH prevented TR inactivation by the peroxidase/H2O2/PTZ+* systems. Production of PTZ+* cation radicals by PTZ peroxidation was essential for TR inactivation. Horseradish peroxidase, leukocyte myeloperoxidase (MPO) and the pseudo-peroxidase myoglobin (Mb) were effective catalysts of PTZ+* production. Promazine, thioridazine, chlorpromazine, propionylpromazine prochlorperazine, perphenazine and trimeprazine were effective constituents of the HRP/H2O2 /PTZ system. The presence of substituents at the PTZ nucleus position 2 exerted significant influence on PTZ activity, as shown by the different effects of 2-trifluoromethyl and 2-H or 2-chlorophenothiazines. The PTZ+* cation radicals disproportionation regenerated the non-radical PTZ molecule and produced the PTZ sulfoxide that was inactive on TR. Thiol compounds including GSH interacted with PTZ+* cation radicals transferring an electron from the sulfide anion to the PTZ+*, thus nullifying the PTZ+* biological and chemical activities.
Subject(s)
Enzyme Inhibitors/pharmacology , NADH, NADPH Oxidoreductases/drug effects , Reactive Oxygen Species/metabolism , Animals , Cations, Monovalent , Electron Spin Resonance Spectroscopy , Free Radicals , Horseradish Peroxidase/metabolism , Horseradish Peroxidase/pharmacology , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Kinetics , Myoglobin/metabolism , Myoglobin/pharmacology , Peroxidase/metabolism , Peroxidase/pharmacology , Phenothiazines/metabolism , Phenothiazines/pharmacology , Recombinant Proteins/antagonists & inhibitors , Structure-Activity Relationship , Sulfhydryl Compounds/pharmacology , Time Factors , Trypanosoma cruziABSTRACT
Peroxidase/H2O2/phenothiazine systems produced irreversible inhibition (inactivation) of Trypanosoma cruzi trypanothione reductase (TR). The enzyme inactivation depended on (a) the incubation time of TR with the peroxidase/H2O2/phenothiazine system; (b) the peroxidase nature and (c) the phenothiazine structure. With the more effective peroxidase/H2O2/phenothiazine systems, TR inactivation kinetics presented a relatively fast initial phase, lasting for about 10 min, in which most of the enzyme activity disappeared. This phase was followed by a slower one and, after 30 min incubation, TR was totally inactivated. Three peroxidases were assayed as catalysts of TR inactivation: the horseradish peroxidase (HRP), leukocyte myeloperoxidase (MPO) and modified myoglobin (Mb). Under comparable experimental conditions, the peroxidase system activity decreased in the given order. With HRP systems, 10 microM Thioridazine (TRDZ), Promazine (PZ), Trimeprazine (TMPZ), Prochlorperazine (PCZ), Propionylpromazine (PPZ), Chlorpromazine (CPZ) and Perphenazine (PFZ), produced 95-100 inactivation of TR. With the MPO/H2O2 systems, PZ. TRDZ and TMPZ were the most effective. Under similar experimental condition, the Mb/H2O2/PZ,/TMPZ, /TRDZ and CPZ systems effectively inactivated TR. The presence of alkylamino, piperazinyl, or piperidinyl groups in PTZ N atom (position 10) and -Cl, -CF3, -SCH3, COCH2CH3 and -CN in position C2 exerted significant influence on phenothiazine activity. Glutathione (GSH) prevented TR inactivation by the HRP/H2O2/PZ and MPO/H2O2/PZ systems. The HRP/H2O2 and MPO/H2O2/phenothiazines systems generated the corresponding cationic radicals (FTZ.+) the stability of which was limited by their conversion into phenothiazine-sulfoxides (PTZ-SO). The latter ones were inactive on TR. GSH rapidly reacted with PTZ+.; thus producing cation radical detoxication. These reactions fit in well with GSH protection of TR against the peroxidase/H2O2/phenothiazine system, as well as with the FTZ.+ role in phenothiazine cytotoxicity.(AU)
Subject(s)
Comparative Study , Animals , Antiprotozoal Agents/pharmacology , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Phenothiazines/pharmacology , Protozoan Proteins/antagonists & inhibitors , Trypanosoma cruzi/enzymology , Antiprotozoal Agents/chemistry , Cations/pharmacology , Free Radicals , Glutathione/metabolism , Hydrogen Peroxide/pharmacology , Kinetics , Molecular Structure , Oxidation-Reduction , Peroxidases/pharmacology , Phenothiazines/chemistry , Recombinant Fusion Proteins/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Peroxidase/H2O2/phenothiazine systems produced irreversible inhibition (inactivation) of Trypanosoma cruzi trypanothione reductase (TR). The enzyme inactivation depended on (a) the incubation time of TR with the peroxidase/H2O2/phenothiazine system; (b) the peroxidase nature and (c) the phenothiazine structure. With the more effective peroxidase/H2O2/phenothiazine systems, TR inactivation kinetics presented a relatively fast initial phase, lasting for about 10 min, in which most of the enzyme activity disappeared. This phase was followed by a slower one and, after 30 min incubation, TR was totally inactivated. Three peroxidases were assayed as catalysts of TR inactivation: the horseradish peroxidase (HRP), leukocyte myeloperoxidase (MPO) and modified myoglobin (Mb). Under comparable experimental conditions, the peroxidase system activity decreased in the given order. With HRP systems, 10 microM Thioridazine (TRDZ), Promazine (PZ), Trimeprazine (TMPZ), Prochlorperazine (PCZ), Propionylpromazine (PPZ), Chlorpromazine (CPZ) and Perphenazine (PFZ), produced 95-100 inactivation of TR. With the MPO/H2O2 systems, PZ. TRDZ and TMPZ were the most effective. Under similar experimental condition, the Mb/H2O2/PZ,/TMPZ, /TRDZ and CPZ systems effectively inactivated TR. The presence of alkylamino, piperazinyl, or piperidinyl groups in PTZ N atom (position 10) and -Cl, -CF3, -SCH3, COCH2CH3 and -CN in position C2 exerted significant influence on phenothiazine activity. Glutathione (GSH) prevented TR inactivation by the HRP/H2O2/PZ and MPO/H2O2/PZ systems. The HRP/H2O2 and MPO/H2O2/phenothiazines systems generated the corresponding cationic radicals (FTZ.+) the stability of which was limited by their conversion into phenothiazine-sulfoxides (PTZ-SO). The latter ones were inactive on TR. GSH rapidly reacted with PTZ+.; thus producing cation radical detoxication. These reactions fit in well with GSH protection of TR against the peroxidase/H2O2/phenothiazine system, as well as with the FTZ.+ role in phenothiazine cytotoxicity.
Subject(s)
Animals , Antiprotozoal Agents , NADH, NADPH Oxidoreductases , Phenothiazines , Protozoan Proteins/antagonists & inhibitors , Trypanosoma cruzi , Antiprotozoal Agents , Cations , Free Radicals , Glutathione , Kinetics , Molecular Structure , Oxidation-Reduction , Peroxidases , Hydrogen Peroxide/pharmacology , Phenothiazines , Recombinant Fusion Proteins/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
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.
ABSTRACT
Peroxidase/H2O2/phenothiazine systems produced irreversible inhibition (inactivation) of Trypanosoma cruzi trypanothione reductase (TR). The enzyme inactivation depended on (a) the incubation time of TR with the peroxidase/H2O2/phenothiazine system; (b) the peroxidase nature and (c) the phenothiazine structure. With the more effective peroxidase/H2O2/phenothiazine systems, TR inactivation kinetics presented a relatively fast initial phase, lasting for about 10 min, in which most of the enzyme activity disappeared. This phase was followed by a slower one and, after 30 min incubation, TR was totally inactivated. Three peroxidases were assayed as catalysts of TR inactivation: the horseradish peroxidase (HRP), leukocyte myeloperoxidase (MPO) and modified myoglobin (Mb). Under comparable experimental conditions, the peroxidase system activity decreased in the given order. With HRP systems, 10 microM Thioridazine (TRDZ), Promazine (PZ), Trimeprazine (TMPZ), Prochlorperazine (PCZ), Propionylpromazine (PPZ), Chlorpromazine (CPZ) and Perphenazine (PFZ), produced 95-100
inactivation of TR. With the MPO/H2O2 systems, PZ. TRDZ and TMPZ were the most effective. Under similar experimental condition, the Mb/H2O2/PZ,/TMPZ, /TRDZ and CPZ systems effectively inactivated TR. The presence of alkylamino, piperazinyl, or piperidinyl groups in PTZ N atom (position 10) and -Cl, -CF3, -SCH3, COCH2CH3 and -CN in position C2 exerted significant influence on phenothiazine activity. Glutathione (GSH) prevented TR inactivation by the HRP/H2O2/PZ and MPO/H2O2/PZ systems. The HRP/H2O2 and MPO/H2O2/phenothiazines systems generated the corresponding cationic radicals (FTZ.+) the stability of which was limited by their conversion into phenothiazine-sulfoxides (PTZ-SO). The latter ones were inactive on TR. GSH rapidly reacted with PTZ+.; thus producing cation radical detoxication. These reactions fit in well with GSH protection of TR against the peroxidase/H2O2/phenothiazine system, as well as with the FTZ.+ role in phenothiazine cytotoxicity.