Remembering a microbiologist: Alfredo Sordelli (1891-1967).
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Argentina↗
Biomedical subjects
A O Stoppani
Publications and source records attributed to A O Stoppani.
Trypanosoma cruzi trypanothione reductase is inactivated by peroxidase-generated phenothiazine cationic radicals.
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.
Animals↗
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.
Adenosine Triphosphate↗
[Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use].
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.
Animals↗
Synthesis and antitrypanosomal evaluation of E-isomers of 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives. structure-activity relationships.
Several novel semicarbazone derivatives were prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde and semicarbazides bearing a spermidine-mimetic moiety. All derivatives presented the E-configuration, as determined by NMR-NOE experiments. These compounds were tested in vitro as potential antitrypanosomal agents, and some of them, together with the parent compounds, 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives, were also evaluated in vivo using infected mice. Structure-activity relationship studies were carried out using voltammetric response and lipophilic-hydrophilic balance as parameters. Two of the compounds (1 and 3) displayed the highest in vivo activity. A correlation was found between lipophilic-hydrophilic properties and trypanocidal activity, high R(M) values being associated with low in vivo effects.
Aldehydes↗
Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by myeloperoxidase-generated "reactive species".
Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. With MPO/H2O2/NaCl, LADH lipoamide reductase and diaphorase activities significantly decreased as a function of incubation time. Iodide, bromide, thiocyanide and chloride effectively supplemented the MPO/H2O2 system, KI and NaCl being the most and the least effective supplements, respectively. LADH inactivation by MPO/H2O2/NaCl and by NaOCl was similarly prevented by thiol compounds such as GSH, L-cysteine, N-acetylcysteine, penicillamine and N-(2-mercaptopropionyl-glycine) in agreement with the role of HOCI in LADH inactivation by MPO/H2O2/NaCl. LADH was also inactivated by MPO/NADH/halide, MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 systems. Catalase prevented the action of the NADH-dependent systems, thus supporting H2O2 production by NADH-supplemented LADH. MPO inhibitors (4-aminobenzoic acid hydrazide, and isoniazid), GSH, L-cysteine, L-methionine and L-tryptophan prevented LADH inactivation by MPO/H2O2/NaNO2. Other MPO systems inactivating LADH were (a) MPO/H2O2/chlorpromazine; (b) MPO/H2O2/monophenolic systems, including L-tyrosine, serotonin and acetaminophen and (c) MPO/H2O2/di- and polyphenolic systems, including norepinephrine, catechol, nordihydroguaiaretic acid, caffeic acid, quercetin and catechin. Comparison of the above effects and those previously reported with pig myocardial LADH indicates that both enzymes were similarly affected by the MPO-dependent systems, allowance being made for T. cruzi LADH diaphorase inactivation and the greater sensitivity of its LADH lipoamide reductase activity towards the MPO/H2O2/NaCl system and NaOCl.
Animals↗
[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.
Animals↗
[Dr. Osvaldo Fustinoni (1909-2000)].
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Journal Article↗
Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds.
Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H2O2/halide, MPO/NADH/halide and MPO/H2O2/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H2O2 system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H2O2/NaCl system. NaOCl and the MPO/H2O2/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H2O2 production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horse-radish peroxidase supplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H2O2/NaNO2 system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated "reactive species" oxidize essential thiol groups at LADH catalytic site.
Amino Acids↗
[The chemotherapy of Chagas disease].
To date, Chagas disease has defied all attempts to develop an efficient and safe chemotherapy. Drugs effective on T. cruzi as trypanocidal agents may be classified as (a) drugs of extensive clinical use: Nifurtimox and Benznidazole; (b) drugs of restricted clinical use: azoles (e.g. Ketoconazole, Econazole; Miconazole); Amphotericin B; Allopurinol, Allopurinol ribosides and Primaquine; (d) drugs effective on T. cruzi and in experimental Chagas disease (murine model): alkyllysophospholipids; 5-amino-imidazole-4-carboxamides; bisbenzyl-isoquinolines; cruzipain (crucein) inhibitors; Gossipol; phenothiazines; d) drugs effective in vitro without other reported effects, acridines, actinomycin D, Crystal Violet (gentian violet), diterpenes (Mikania obtusata); N,N'-dimethyl-2-propen-1-amine, epoxidienthiol carbamates, Fe-chelators, guanyl hydrazones, o-naphthoquinones (beta-lapachone); quinoids (miconidine; tingenone); Olivacine, phenazine methosulfate, phenoxi-phenoxyl drugs, Proadifen, pyridinium azolate betaines, sesquiterpenes (Lychophora sp), sesquiterpene lactones, tetrahydrocarbazoles, DL-alpha-trifluoromethylarginine, triphenylmetane dyes. It is generally agreed that Nifurtimox and Benznidazole (a) are effective on acute Chagas' disease, but may not be effective in the chronic phase; (b) their effect depends on the susceptibility of T. cruzi strains to the drug; (c) they produce adverse effects in patients that may prevent prolonged treatments; they are genotoxic and produce biochemical damage in the mammalian tissues. Redox-cycling of Nifurtimox and Benznidazolee generates "reactive oxygen species" which explain the biological effects. At variance with the mammalian host, T. cruzi is deficient in antioxidant enzymes which are essential to prevent oxidative damage. Azoles are effective inhibitors of T. cruzi growth in vitro and in vivo since they inhibit sterol C14-delta 24(25) demethylase, an enzyme catalysing ergosterol production. Azoles reduce parasitemia and extend the survival of infected mice but do not produce parasitological cure and their clinical effectiveness is questionable. Allopurinol allopurinol ribosides and related compounds inhibit T. cruzi hypoxantine-guanine ribosyl transferase, thus preventing the synthesis of adenylic and guanylic acids and also DNA. They reduce parasitemia and negativize xenodiagnosis but these effects may not be permanent, which invalidates their clinical use. Cysteine-protease inhibitors recognize T. cruzi protease (cruzipain, crucein) active site, thus allowing a covalent linkage with the inhibitor. These peptide inhibitors are effective in acute and chronic murine models. Phenothiazines inhibit trypanothione reductase and a specially favoured fit is a small 2-substitued 2-chloro and 2-trifluoromethyl with a remote hydrophobic patch. The essential phenotiazine nucleus can adopt more than one inhibitory orientation in its binding site. Phenothiazines are promising trypanocidal agents for the treatment of Chagas' disease. The methodology for developing new drugs for the treatment of Chagas' disease is discussed.
Animals↗
[AIDS and the challenge of the emergent diseases. Closing remarks].
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Acquired Immunodeficiency Syndrome↗
[Myeloperoxidase as a factor of oxidative damage of the myocardium: inactivation of dihydrolipoamide dehydrogenase].
Myocardial dihydrolipoamide dehydrogenase (LADH) is inactivated after incubation at 30 degree C, with myeloperoxidase (MPO)-dependent systems. The enzyme inactivation was a function of the pro-oxidant system composition and the time of incubation. The standard inactivating system contained 50 mM KH2PO4-K2HPO44, pH 7.4, 0.5-1.0 muM LADH, and pro-oxidant system. After 30 or 60 min of incubation with the MPO/H2O2/NaCl system, LADH inactivation was 64 and 87%, respectively (Figure 1). In the absence of NaCl, inactivation values were 9 and 27%, respectively, whereas in the absence of MPO the inactivation values were 4.0 and 11%, respectively (Figure 1). Under similar experimental conditions, sodium hypochlorite significantly inactivated LADH, thus supporting the role of hipochlorous acid as agent of the MPO/H2O2/CINa system. With the MPO/H2O2/Kl, MPO/H2O2/SCN or the MPO/H2O2/NaNO2 systems LADH inactivation depended on the anion nature, 1-being the most effective (Figure 2). NaNo2 effectively replaced halides as pro-oxidant (Figure 3). The MPO/NADH/halide systems, where NADH replaced H2O2, also inactivated LADH. Native (not denatured) catalase completely prevented the MPO/NADH/Kl system effect (Table 1), in close agreement with H2O2 production by the LADH-catalysed NADH oxidation and the role of H2O2 in LADH inactivation. LADH was also inactivated after incubation with MPO-generated free radicals such as the Chloropromazine and Paracetamol radicals (Table 2). Thiol compounds (Captopril, penicillamine, cysteine, N-acetylcysteine and mercaptopropionylglycine) (Table 3 and Figure 4), as well as taurine, ascorbate (Table 4), GSSG and trypanothione (Figure 5), protected LADH against the MPO-dependent oxidizing systems, and also against NaCIO (Table 4). The summarized observations are discussed in relation to MPO function in free radical production and pathologies such as ischemia-reperfusion injury and inflammation.
Acetaminophen↗
[Physiopathologic effects of nitric oxide and their relationship with oxidative stress].
Nitric oxide (NO.) is produced from L-arginine, as result of a reaction catalyzed by the enzyme nitric oxide synthase (NOS). The reaction is the sole source of NO. in animal tissues. NO. can control physiological processes (or systems) such as (a) blood pressure; (b) relaxation of arterial smooth muscle; (c) platelet aggregation and adhesion; (d) neurotransmission; (e) neuroendocrine secretion. NO. contributes to the killing of pathogenic microorganisms and tumoral cells by phagocytes. NO. reacts with superoxide anion thus producing peroxynitrite, a cytotoxic ion capable of destroying many biological targets. The superoxide/peroxinitrite balance determines the ONOO- production and, accordingly, is essential for the development of hypertension, atherosclerosis, neurodegenerative diseases, viral infections, ischemia-reperfusion injury, and cancer.
Antioxidants↗
Inactivation of yeast glutathione reductase by Fenton systems: effect of metal chelators, catecholamines and thiol compounds.
Oxygen radical generating systems, namely, Cu(II)/ H2O2, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H2O2/catecholamine and Cu(II)/H2O2/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H2O2 enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H2O2 dependent on Cu(II) and H2O2 concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO. scavengers prevented GR inactivation by Cu(II)/H2O2 and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropionylglycine and penicillamine enhanced the effect of Cu(II)/H2O2 in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H2O2 effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and trypanothione disulfide effectively protected GR against Cu(II)/H2O2 inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS.+. GR inactivation by the systems assayed correlated with their capability for HO. radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.
Catecholamines↗
Redox cycling of o-naphthoquinones in trypanosomatids. Superoxide and hydrogen peroxide production.
beta-Lapachone and structurally related lipophilic o-naphthoquinones, namely, CG 8-935, CG 9-442, CG 10-248 and mansonones A, C, E, and F, were investigated for redox cycling, production of reactive oxygen species, and cytotoxicity in the trypanosomatids Crithidia fasciculata and Leptomonas seymouri. Structural analysis of the assayed quinones indicated that a tricyclic structure, including a naphthalene ring, a 1,2b or 1,8bc pyran ring, and two ortho-carbonyl groups were required for quinone activities. The contribution of oxygen radical production to quinone cytotoxicity was supported by: (a) spectroscopic observation of quinone redox cycling; (b) production of the semiquinone radical; (c) H2O2 and O2- production; (d) the effect of beta-lapachone on thiol pools in C. fasciculata; (e) the effect of quinones on cell respiration; (f) superoxide dismutase inactivation after incubation of C. fasciculata with CG 8-935; and (g) the effect of quinones on cell growth.
Animals↗
Redox cycling of beta-lapachone and related o-naphthoquinones in the presence of dihydrolipoamide and oxygen.
Lipophilic o-naphthoquinones (beta-lapachone, CG 8-935, CG 9-442, CG 10-248, and mansonones A, C, E, and F), catalyze the oxidation of dihydrolipoamide (DHLA) by oxygen, whereas p-naphthoquinones (alpha-lapachone and menadione) are scarcely active. The greatest effects corresponded to beta-lapachone and its analogues. Quinol production was demonstrated by (a) the absorption spectrum of the reduced quinone, and (b) the effect of pH variation on the rate of quinone-catalyzed DHLA oxidation. Superoxide dismutase (SOD) inhibited the rate of cytochrome c reduction and decreased the apparent rate of oxygen consumption by several DHLA/o-naphthoquinone systems. SOD also inhibited the rate of quinol oxidation by oxygen, after quinone reduction by a stoichiometric amount of DHLA. Catalase enhanced the effect of SOD, but in its absence catalase was inactive. It is concluded that quinone-catalyzed oxidation of DHLA implies a free-radical mechanism in which the quinol and superoxide radicals play an essential role.
Hydrogen-Ion Concentration↗
Catecholamines enhance dihydrolipoamide dehydrogenase inactivation by the copper Fenton system. Enzyme protection by copper chelators.
Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II)/H2O2 (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H2O2 and CA concentrations. Similar inhibitions were obtained with the assayed CAs and o-diphenols. CAs enhanced HO. radical production by Cu(II)/H2O2, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipoamide, dihydrolipoic acid, DL-dithiothreitol, GSSG, trypanothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H2O2/CAs systems. Denatured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH increased and Captopril inhibited epinephrine oxidation by Cu(II)/H2O2 and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO. from H2O2 by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the enzyme active site by site-specifically generated HO. radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.
3,4-Dihydroxyphenylacetic Acid↗
Inactivation of heart dihydrolipoamide dehydrogenase by copper Fenton systems. Effect of thiol compounds and metal chelators.
Copper Fenton systems (Cu(II)/H2O2 and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H2O2 treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD+ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H2O2. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 microM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H2O2. Allopurinol provided partial protection against Cu(II)/H2O2. Ethanol, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H2O2 or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H2O2. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.
Animals↗