Yolanda Repetto

STUDIES ON THE METABOLISM OF ECHINOCOCCUS GRANULOSUS. VII. REACTIONS OF THE TRICARBOXYLIC ACID CYCLE IN E. GRANULOSUS SCOLICES.

Abstract 1. 1. E. granulosus intact scolices as well as cell-free preparations oxidize several Krebs acid cycle intermediates. Cell-free preparations contain condensing enzyme, a TPN- and DPN-dependent isocitric dehydrogenase, succinic, malic and α-ketoglutaric dehydrogenases, as well as fumarase, aconitase, pyruvic oxidase and α-carboxylase. 2. 2. Phosphoenolpyruvic, carboxydismutase and probably “malic” enzyme are involved in CO 2 -fixation reactions in E. granulosus . 3. 3. Acetate is activated by aceto-CoA-kinase. No evidence was found for acetokinase and transacetylase, although the extracts contained an active acetyl-phosphatase. The glyoxylate cycle seems to be ruled out since neither isocitriatase nor malate synthetase was present in homogenates or acetone powder extracts. 4. 4. C 14 -labeled carbon dioxide is utilized by intact scolices for the synthesis of Krebs acid cycle intermediates, protein, lipids and phospholipids, nucleic acids and polysaccharides. Most of the C 14 is fixed into succinic acid, which is excreted into the medium. Acetate-2-C 14 is also utilized for the synthesis of various cellular materials, including polysaccharides. 5. 5. The above findings, together with the labeling patterns from C 14 O 2 -fixation of the amino acids obtained from protein hydrolysates as well as of the Krebs acid cycle intermediates, are considered as an indication of the presence of the Krebs acid cycle in E. granulosus . 6. 6. A C 3 -C 1 -type of C 14 O 2 -fixation with the formation of oxalacetate which is turn reduced into succinate via malate-fumarate is advanced as a possible mechanism for the production of succinate.

Studies on the metabolism of Echinococcus granulosus—VIII. The pathway to succinate in E. Granulosus scolices☆

Abstract 1. 1. The mechanism of succinate production has been investigated in E. granulosus scolices. 2. 2. The evidence obtained is in agreement with the formation of pyruvate by glycolysis, followed by CO2-fixation into pyruvate via “malic enzyme” and the reduction of the carboxylation product into succinate. 3. 3. The pentose phosphate pathway contributes, although to a minor extent, to succinate formation.

Glutathione and trypanothione in several strains of Trypanosoma cruzi: effect of drugs.

Glutathione (GSH), trypanothione (T(SH)2) and glutathionyl spermidine (GSH-SP) concentrations were determined in the Tulahuén and LQ strains and the DM 28c clone of Trypanosoma cruzi. The concentrations of GSH, T(SH)2 and GSH-SP, expressed as nmol of GSH per g of parasite fresh weight, were 60.1, 397.8 and 103.9, respectively, for the Tulahuén strain. For the DM 28c clone, the values were 113.9, 677.9 and 164.1, respectively, and for the LQ strain they were 199.1, 1100.5 and 55.3, respectively. When the parasites were treated with 10 microM nifurtimox or 50 microM benznidazole for 2 h, the concentrations of all three reduced thiols decreased strongly. The total amount of T(SH)2 decreased by more than 50%. Treatment of the parasites with 5 mM buthionine sulfoximine, an inhibitor of GSH synthesis, for 6 h diminished the concentrations of the reduced thiols by between 27% and 53% with respect to the controls. Cyclohexylamine, an inhibitor of spermidine synthesis, decreased the concentrations of T(SH)2 and GSH-SP but not that of GSH. It is possible to conclude from this study that trypanothione is the most important thiol involved in the detoxication of nifurtimox and benznidazole in T. cruzi and that electrophilic reduced metabolites of both drugs are most probably conjugated with GSH, T(SH)2 and GSH-SP, thus decreasing their concentrations. GSH biosynthesis is an important drug target.

t-Butyl-4-hydroxyanisole, a novel respiratory chain inhibitor: Effects on Trypanosoma cruzi epimastigotes

t-Butyl-4-hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level ofcytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.t‐Butyl‐4‐hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level ofcytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.

Involvement of thiol metabolism in resistance to glucantime in Leishmania tropica

Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.

Effects and mode of action of 1,4-naphthoquinones isolated from Calceolaria sessilis on tumoral cells and Trypanosoma parasites

The naphthoquinones 2-hydroxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-1), (-)-2,3,3-trimethyl-2-3-dihydronaphtho[2,3-b]furan-4,9-quinone (CS-3), and 2-acetoxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-5) isolated from Calceolaria sessilis were tested against Trypanosoma cruzi epimastigotes, the TA3 tumor cell line and the methotrexate-resistant subline TA3-MTX-R. Naphthoquinone CS-3 was the most active; the 50% culture growth inhibition (I50) on T. cruzi (Tulahuén and LQ strain and DM28c clone) was at concentrations ranging from 2.1 to 5.2 mumolar. Also CS-3 inhibited TA3 and TA3-MTX-R culture growth with an I50 of 2.1 and 3.8 mumolar, respectively. Naphthoquinone CS-3 inhibited the respiration of the tumor cells by interfering with the electron transport at some point between NADH and ubiquinone. The respiration of T. cruzi was not inhibited by naphthoquinone CS-3. Naphthoquinone CS-3 produced a temporary increase of oxygen consumption in T. cruzi and tumor cells, suggesting the generation and participation of free radicals.

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.

Effects of some β-carboline alkaloids on intact Trypanosoma cruzi epimastigotes

Several beta-carboline (9H-pyrido-[3,4-b]-indole) alkaloids were evaluated for in vitro trypanosomicidal activity against Trypanosoma cruzi epimastigotes belonging to two different strains (Tulahuén and LQ) showing different sensitivity to nifurtimox. Important differences were observed in the susceptibility of the parasites to these natural substances, with the relatively nifurtimox-resistant LQ strain showing greater sensitivity to the beta-carbolines. Respiratory chain inhibition appears to be a possible determinant of the trypanosomicidal activity of these compounds.

Novel Benzo[1,2-c]1,2,5-Oxadiazole N-Oxide Derivatives as Antichagasic Agents: Chemical and Biological Studies

This research was supported by FONDECYT 1000834, 7040037, 1020095, CSIC (UdelaR), CLEMENTE ESTABLE and TWAS grants. We thank a scholarship for C. Rigol from RELACQ.

Characterization of glutathione s-transferase activity in echinococcus granulosus☆

1. Glutathione S-transferase activity has been characterized in protoscolices of Echinococcus granulosus. 2. The conjugation reaction was activated by treatment of protoscolices with phenobarbital and beta-naphthoflavone. 3. Protoscolices of E. granulosus contained the conjugation cofactor glutathione in the amount of 287 nmol/g fresh tissue. 4. Conjugation may represent an important factor in the resistance of the parasite to drugs.