Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

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)

Diabetes y transporte de calcio en mitocondrias hepaticas / Diabetes and calcium transportation in liver mitochondria

Se aislaron las mitocondrias hepáticas de ratas normales y ratas con diabetes crónica por inyección de estreptozotocina. Se determinó su capacidad de captar Ca2+ en un medio isotónico, con succinato como fuente de energía y Antipirilazo III como indicador de [Ca2+]. Se comprobó que la velocidad de captación fue significativamente menor en las mitocondrias diabéticas. La inhibivión con Ruthenium Red permitió medir la velocidad de eflujo del Ca2+, que resultó significativamente mayor que en las mitocondrias normales. La medida del potencial de membrana con safranina como indicador dio un valor significativamente menor en las mitocondrias diabéticas, en concordancia con la disminución de la captación de Ca2+ (AU)

Menor oxidación de cuerpos cetónicos por mitocondrias de corazón en la diabetes / Decreased oxidation of ketone bodies in heart mitochondrias in diabetes

Ratas diabéticas por inyección de estreptozotocina presentaron concentraciones de 3-hidroxibutirato y acetoacetato en sangre 4,2 y 1,7 veces superiores a las normales, respectivamente. Al mismo tiempo, en las mitocondrias de corazón disminuyó la actividad de las enzimas iniciadoras del metabolismo oxidativo de esos cuerpos cetónicos, a saber, la 3-hidroxibutirato deshidrogenasa (72%) y la succinil-CoA: acetoacetil-CoA (3 - oxoácido - CoA) transferasa (50%). En cambio, la acetoacetil-CoA tiolasa, no varió. La oxidación del 3-hidroxibutirato y el acetoacetato por las mitocondrias enteras de ratas diabéticas, suplementadas con ADP (en estado metabólico "3") disminuyó 42 y 48%, respectivamente, en relación a los testigos normales, no así la oxidación del piruvato o del L-glutamato más L-malato que no varió significativamente. Estas observaciones implican una modificación selectiva de las enzimas correspondientes a los cuerpos cetónicos. La composición lipídica y la depolarización de la fluorescencia del difenil hexatrieno en las mitocondrias diabéticas no presentaron diferencias respecto a las normales, de manera que las variaciones enzimáticas descriptas se pueden atribuir a una síntesis defectuosa de las proteínas mitocondriales (AU)

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)

Efectos del nifurtimox, benznidazol y beta-lapachona sobre el metabolismo del ADN, ARN y proteínas en Trypanosoma cruzi. / [Effects of nifurtimox, benznidazole, and beta-lapachone on the metabolism of DNA, RNA and proteins in Trypanosoma cruzi]

Nifurtimox and benznidazole have trypanostatic actions in vitro and inhibit the incorporation of [3H] thymidine, [3H] uridine and L-[3H] leucine in T. cruzi macromolecules. The effect of nifurtimox may be explained by (a) direct inhibition of nucleic acid biosynthesis, or (b) generation of the oxygen radicals in T. cruzi and therefore, only mechanism (a) should be valid. In order to obtain more information on the action of these drugs on T. cruzi, in the present study we examined the effect of nifurtimox and benznidazole on DNA, RNA and protein turnover in epimastigote (culture) forms of the parasite. Complementary experiments were performed with beta-lapachone that, like nifurtimox, generates oxygen radicals in T. cruzi. Epimastigotes (Tulahuen strain) at the exponential-phase of growth were cultured with [3H] thymidine, [3H] uridine or L-[3H] leucine to label DNA, RNA and protein, respectively. After incubation, the cells were washed free of radioactive precursor, resuspended in fresh medium and reincubated at 30 degrees C with nifurtimox (10 or 100 microM), benznidazole (38 or 380 microM) or beta-lapachone (1.6 or 7.8 microM), for 1-3 hours. Controls were incubated without drug. At one hour time intervals, sampler were taken, washed free of medium and filtered through 0.45 microns Metricel filters. The filters were washed with 10

trichloroacetic acid to remove the acid soluble material, and after drying, the radioactivity incorporated in DNA, RNA and protein was counted with a scintillation counter. The results show that after elimination of the labelled precursors, 3H activity in DNA, RNA and protein decayed as a function of the time of incubation. Nifurtimox, benznidazole and beta-lapachone, stimulated in all cases they decay of the incorporated radioactivity. Calculation of [quot ]half-life[quot ] values for DNA, RNA and protein(s) indicated that nifurtimox and beta-lapachone exerted their greatest effects on DNA while benznidazole increased the decay of DNA, RNA and protein to about the same extent. Taking into account the effects of nifurtimox and beta-lapachone on DNA stability, specific lesions (single-strand breaks) were investigated in DNA from control, nifurtimox, benznidazole or beta-lapachone treated epimastigotes. The number of single-strand breaks was (per 10(6)b) 25 with 100 microM nifurtimox, 1.4 with 380 microM benznidazole and 45 with 7.8 microM g-lapachone. Interestingly enough, after reincubation of nifurtimox-damaged epimastigotes in fresh medium for 24 hours, recovery of DNA lesions could be observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Existencia de dos sitios de fosforilación oxidativa en Trypanosoma cruzi. / [Existence of 2 sites of oxidative phosphorylation in Trypanosoma cruzi]

Phosphorylating mitochondrial preparations were obtained from T. cruzi culture (epimastigote) forms by grinding the cells with glass - beads and differential centrifugation. Using ADP as phosphate acceptor and succinate, L-malate or ascorbate + tetramethyl-p-phenylenediamine (TMPD) as oxidizable substrates, the respiratory control (R.C.) values were (mean +/- S.D.; n = 4, in parenthesis, the substrate): 2.8 +/- 0.10 (succinate): 2.3 +/- 0.13 (L-malate) and 2.0 +/- 0.12 (ascorbate + TMPD). The ADP:O values were 1.68 +/- 0.08, 1.42 +/- 0.08 and 0.66 +/- 0.66 +/- 0.12, respectively. The uncoupler CCCP stimulated substrate oxidation somewhat more than ADP. Succinate oxidation was by malonate and also by oxaloacetate but the latter was effective only after sonicating the mitochondrial preparation. The mitochondrial membranes oxidized also NADH but this oxidation was not subjected to control by the phosphate acceptor. Our results support the existence of two energy-conserving sites in T. cruzi respiratory chain (sites 2 and 3) and confirm previous observations by Stoppani et al. (Mol. Biochem. Parasitol. 2:3-21, 1980) with intact epimastigotes.

Muerte celular programada y apoptosis. Función de las mitocondrias. / [Programmed cell death and apoptosis. The role of mitochondria]

Physiological cell death and apoptosis are natural processes genetically programmed, subjected to control by complex molecular mechanisms which elucidation is of particular interest for biology and medicine. Mitochondria play an essential role in physiological cell death and apoptosis. Apoptogenic effects develop in three phases, namely: (a) premitochondrial; (b) mitochondrial and (c) post-mitochondrial. During the first phase, apoptogenic signals (genotoxic agents, oxygen free radicals, corticoids, antibodies, etc.) interact with cell receptors activating specific mechanisms including thiol dependent proteases (caspases). As a consequence of those signals, mitochondrial damage results (membrane permeabilization, collapse of the membrane potential, swelling, membrane disruption, inhibition of electron transfer and oxidative phosphorylation). Other consequences of the mitochondrial disruption are the enhancement of free radical production and the exit of cytochrome c, caspases and endonucleases to the cytosol. During the third phase of apoptosis, free radicals and activated enzymes attack the cell protein structure and ADN, thus causing cell death. The mitochondrial regulation of apoptosis is controlled by the mitochondrial transitory permeability pore (MTPP) which is constituted by caspases, hexokinases, cytochrome c, ATP and ADP. MTPP is subjected to control by apoptogenic or antiapoptogenic agents which open or close it, according to their structure and the cell metabolic conditions. Uncontrolled opening of MTPP determines a massive exit of mitochondrial apoptogenic factors which in the cytosol and the nucleus exert their apoptogenic effects, thus producing cell death. MTPP can be modified by drugs with potential therapeutic actions thus opening interesting therapeutic possibilities. The role of apoptosis in pathologies such as degenerative diseases of the nervous system, autoimmunity diseases, SIDA and cancer is discussed.

Inactivación de la dihidrolipoamida deshidrogenasa de miocardio por Cu(II) y peróxido de hidrógeno. / [Inactivation of myocardial dihydrolipoamide dehydrogenase by Cu(II) and hydrogen peroxide]

The inactivation of pig-heart dihydrolipoamide (LipDH) by oxy-radicals generated by Cu(II), supplemented or not with hydrogen peroxide (Fenton system-Cu(II): SF-Cu(II)) or ascorbate (Cu(II)--Asc), was studied. The reagents concentrations used were 2.5-10 microM Cu(II): 3.0 mM H2O2, and 0.5 mM ascorbate. After 5 minutes incubation at 30 degrees, LipDH activity was measured as described by Gutiérrez Correa and Stoppani (Reference 13). As a result of peroxide effect, LipDH lipoamide reductase activity decreased in most cases by 83-98

(with the SF-Cu(II) and Cu(II)-Asc system) or 46-53

with Cu(II) only. The enzyme diaphorase activity increased several-fold (Table 1), thus showing a site-specific damage of LipDH thiols. NAD+, dihydrolipoamide, GSSG, CAPTO-PRIL, metal chelators (L-histidine, bathocuproine, EDTA, DETAPAC), trypanothione and allopurinol) protected LipDH from inactivation by SF-Cu(II) (Tables 2, 4-6). The same compounds, GSH, dithiothreitol, N-acetylcysteine, mercaptopropionylglycine and DL-penicillamine protected the enzyme from inactivation by Cu(II) (Tables 2, 4-6). L-cysteine only protected from Cu(II), to a limited degree (Table 4). Compounds protecting LipDH did not reactivate the inactivated enzyme (Table 7). NADH (Table 2), OH-DOPAMINE, DOPA, dihydroxy-phenylacetic acid (DOPAC) and catechol (Table 8) enhanced LipDH inactivation by the SF-Cu(II) but not by Cu(II), except OH-dopamine. ATP and ADP enhanced LipDH inactivation by Cu(II), but not by SF-Cu(II) (Table 3). HO scavengers (benzoate, mannitol, ethanol) and superoxide dismutase did not prevent LipDH inactivation by Cu(II) and H2O2. Catalase protected but its action was not related to its catalytic activity (Table 9). LipDH inactivation by oxygen radicals and its modification by therapeutic agents are discussed in the context of the physiopathology of heart injury after post-ischemic reoxygenation.

Efectos del nifurtimox, benznidazol y beta-lapachona sobre el metabolismo del ADN, ARN y proteínas en Trypanosoma cruzi / [Effects of nifurtimox, benznidazole, and beta-lapachone on the metabolism of DNA, RNA and proteins in Trypanosoma cruzi]

Nifurtimox and benznidazole have trypanostatic actions in vitro and inhibit the incorporation of [3H] thymidine, [3H] uridine and L-[3H] leucine in T. cruzi macromolecules. The effect of nifurtimox may be explained by (a) direct inhibition of nucleic acid biosynthesis, or (b) generation of the oxygen radicals in T. cruzi and therefore, only mechanism (a) should be valid. In order to obtain more information on the action of these drugs on T. cruzi, in the present study we examined the effect of nifurtimox and benznidazole on DNA, RNA and protein turnover in epimastigote (culture) forms of the parasite. Complementary experiments were performed with beta-lapachone that, like nifurtimox, generates oxygen radicals in T. cruzi. Epimastigotes (Tulahuen strain) at the exponential-phase of growth were cultured with [3H] thymidine, [3H] uridine or L-[3H] leucine to label DNA, RNA and protein, respectively. After incubation, the cells were washed free of radioactive precursor, resuspended in fresh medium and reincubated at 30 degrees C with nifurtimox (10 or 100 microM), benznidazole (38 or 380 microM) or beta-lapachone (1.6 or 7.8 microM), for 1-3 hours. Controls were incubated without drug. At one hour time intervals, sampler were taken, washed free of medium and filtered through 0.45 microns Metricel filters. The filters were washed with 10

trichloroacetic acid to remove the acid soluble material, and after drying, the radioactivity incorporated in DNA, RNA and protein was counted with a scintillation counter. The results show that after elimination of the labelled precursors, 3H activity in DNA, RNA and protein decayed as a function of the time of incubation. Nifurtimox, benznidazole and beta-lapachone, stimulated in all cases they decay of the incorporated radioactivity. Calculation of [quot ]half-life[quot ] values for DNA, RNA and protein(s) indicated that nifurtimox and beta-lapachone exerted their greatest effects on DNA while benznidazole increased the decay of DNA, RNA and protein to about the same extent. Taking into account the effects of nifurtimox and beta-lapachone on DNA stability, specific lesions (single-strand breaks) were investigated in DNA from control, nifurtimox, benznidazole or beta-lapachone treated epimastigotes. The number of single-strand breaks was (per 10(6)b) 25 with 100 microM nifurtimox, 1.4 with 380 microM benznidazole and 45 with 7.8 microM g-lapachone. Interestingly enough, after reincubation of nifurtimox-damaged epimastigotes in fresh medium for 24 hours, recovery of DNA lesions could be observed.(ABSTRACT TRUNCATED AT 400 WORDS)

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.(AU)

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 AM 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 (AU)

Influencia de naftoquinona-iminas sobre la producción de peróxido de hidrógeno por Crithidia fasciculata y Leptomonas seymouri. / [Effect of naphthoquinone-imines on the production of hydrogen peroxide by Crithidia fasciculata and Leptomonas seymouri]

It has been synthesized a series of 4-aminoisoxazol-1,2-naftoquinones, which inhibit growth and DNA synthesis in Trypanosoma cruzi. This effect was related to quinone induction of oxyradical generation. N-(5-methyl-3-isoxazolyl)-4-amino-1,2-naftoquinone and 2-hydroxy-N-(3,5-dimethyl-1-4-isoxazolyl)-1,4-naftoquinone-4 -imine inhibited also growth of Crithidia fasciculata and Leptomonas symouri, two organisms that may prove useful for the assay of trypanocidal drugs. In order to establish the role of oxyradicals for quinone-imine toxicity, the latter redox-cycling was demonstrated by a) reversible change in their spectra; b) reduction under anaerobic conditions, and c) quinol oxidation by oxygen. Production of H2O2, measured by the microperoxidase method, was maximal with N-(5-methyl-3-isoxazolyl)-4-amine-1,2-naftoquinone (IVD) (0.29-0.26 nmol/min/mg of cell protein), either with C. fasciculata or L. seymouri. Lesser, though significant activities were obtained with 2-hydroxy-N-(5-methyl-3-isoxazolyl)-1,4-naftoquinone-4-imine (IIID) and 2-hydroxy-N-(3-methyl-5-isoxazolyl)-1,4-naftoquinone-4-imine (IIIE) whereas IIIA and IIIC were inactive. O2-. generation, measured by the adrenochrome method, was induced by naftoquinone-imines with a hydroxyl group at C-2 (IIIE, 2-hydroxy-N-(3,5-dimethyl-4-isoxazolyl)-1,4-naftoquinone-4-i mine (IIIC) and 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naftoquinone-4-i mine (IIIA), but no by IVD, with a carbonyl group at C-2. Measurement of catalase and superoxide dismutase in both organisms yielded significant activities, but ascorbate peroxidase, guaiacol peroxidase and bencidine peroxidase were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Sobre efectos indeseables del benznidazol y el nifurtimox. / Undesirable effects of benznidazole and nifurtimox

La adicion de benznidazol a microsomas de higado de rata, en presencia de NADPH produjo incremento en el consumo de oxigeno y la formacion de anion superoxido y peroxido de hidrogeno ("oxigeno activo"). Estos efectos fueron inhibidos por NADP y p-cloromercuribenzoato, inhibidores que demuestran la intervencion de la NADPH-citocromo P-450(c) reductasa microsomal en la reduccion univalente del benznidazol. En cambio la metyrapone y el SKF-525-A no inhibieron los efectos metabolicos indicados, lo que excluye al citocomo P-450 como intermediario en la reduccion microsomal del benznidazol. Las concentraciones de benznidazol capaces de inducir la formacion de "oxigeno activo" fueron 10 veces mayores que las correspondientes al nifurtimox (Docampo y col. Arch. Biochem Biophys 207:316, 1981), lo que concuerda con la menor toxicidad clinica del benznidazol (Laplume y Col.Medicina (Bs Aires 42: 223, 1982). En contraste con el nifurtimox (Docampo, Stoppani Arch Biochem Biphys 197: 317, 1979), el benznidazol no indujo la formacion de anion superoxido y peroxido de hidrogeno por el sistema microsomal de T. cruzi

Acción de beta y alpha-lapachona sobre la producción de h2o2 y el crecimiento de Trypanosoma cruzi / [Effect of beta and alpha-lapachone on the production of H202 and on the growth of Trypanosoma cruzi]

Addition of beta-lapachone to the epimastigote (culture) form of Trypanosoma cruzi, suspended in saline, buffered-isotonic medium (pH 7.2), determined the appearance of large amounts of H2O2 in the suspension medium, as measured spectrophotometrically by formation of the H2O2 horse radish peroxidase complex. Under similar conditions, alpha-lapachone did not induce H2O2 formmation. Using NADH as electron donor, beta-lapachone (not alpha-lapachone) increased significantly the rate of H2O2 generation by epimastigote homogenates and the same occurred with NADPH, although in a reduced extent. Similar results were obtained with the isolated mitochondrial and microsomal fractions although with the latter NADPH was more effective than NADH as electron donor for beta-lapachone reduction and peroxide generation. The distribution of peroxide generation in epimastigote fractions would indicate that about 92

of the beta-lapachone dependent formation of peroxide occurred in the mitochondria, and 8

in the endoplasmic reticulum. The growth of epimastigotes was inhibited 95

by 1 microgram/ml beta-lapachone, a concentration that determined maximal rate of H2O2 production. Since H2O2 and other intermediates of oxygen reduction such as O2- (superoxide anion) and OH (hydroxyl radical) are lethal to cells and tissues, it is possible that the effect of beta-lapachone on T. cruzi proliferation in vitro was mediated by H2O2 and related free radicals.

Inactivación de la tripanotiona reductasa de Trypanosoma cruzi por radicales libres catiónicos de fenotiazinas / Inactivation of Trypanosoma cruzi trypanothione reductase by phenothiazine cationic free radicals

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)

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.(AU)

Acción inhibitoria de sistemas Fenton sobre la topoisomerasa I de Trypanosoma cruzi y Crithidia fasciculata / Effect of Fenton systems on Trypanosoma cruzi and Crithidia fasciculata topoisomerase I

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)

Inhibición de la glutation reductasa por nifurtimox y otros derivados del 5-nitrofurano / Glutathione reductase inhibition by nifurtimox and related nitrofurans

Derivados del 5-nitrofurano portadores de heterociclos nitrogenados insaturados inhibieron la GR de hígado, corazón y riñoz de rata, con mayor eficiencia que portadores de hetereciclos saturados o de grupos de estructura acíclica , como el nifurtimox, la nitrofurazona y el ácido 5 nitro-2-furoico. La inhibición muestra cinética acompetitiva respecto a los sustratos NADPH y GSSG y no implica un ciclo redox del grupo nitro o diversión de electrones al oxígeno, con formación de oxi-radicales. Ell 2-nitroimidazol y su derivado el benznidazolm el 5-nitroindol y el cloranfenicol (derivado del nitrobencilo), no inhibieron la GR. La GP, enzima asociada fisiológicamente a la GR para la desintoxicación de hidroperóxidos no fue inhibida por los nitrofuranos activos sobre la GR, cuando la disponibilidad de GSH fue suficiente para la actividad de la GP. Cuando la actividad de la GR fue limitante de la concentración de GSH, la actividad GP del sistema fue inhibida (AU)

Efecto de quinonas y nitrofuranos sobre Trypanosoma mega y Crithidia fasciculata. / [Effect of quinones and nitrofurans on Trypanosoma mega and Crithidia fasciculata]

Demonstration of trypanocidal effects in vitro is a first step for the development of new antichagasic drugs. In order to obtain an experimental model allowing the pre-screening of potential trypanocides for Trypanosoma cruzi in a short time and under safe conditions, the trypanosomatids T. mega and C. fasciculata were assayed for their response to a) compounds known for their action on T. cruzi, and b) compounds not tested before on the latter. The drugs were assayed on the organisms growth in a liquid culture medium, cell multiplication being measured by the medium turbidity increase, using a photoelectric colorimeter previously calibrated with cell suspensions of known concentration. A series of quinones (Lapachones and related compounds), naftoquinone-imines, benzoquinones (perezone and dihydroperezone), a quinol (miconidine) and several nitrofurans, including nifurtimox and (5-nitro-2-furfurylidene)-amino (NF-group) derivatives, inhibited the flagellates growth, specially T. mega, with half-maximal inhibitory concentrations lesser than 5.0 microM, for the most active compounds. T. mega response to nifurtimox, NF-derivatives and beta-lapachone was in close agreement with that of T. cruzi. Cultures of T. mega in the presence of NF-pyrazole, NF-indazoles and NF-imidazole but not nifurtimox, showed irreversible damage since, after re-incubation in fresh medium without inhibitor, these cells grew significantly less than their corresponding controls. Similar effects were observed in C. fasciculata, with beta-lapachone and one naftoquinone-imine. Our results qualify T. mega as an adequate experimental model for the assay of antichagasic agents, as C. fasciculata and T. brucei brucei do for the african trypanosomes.