J. Aldunate

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.

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.

Trypanocidal effect of boldine and related alkaloids upon several strains of Trypanosoma cruzi

The alkaloids boldine, glaucine, predicentrine, apomorphine, coclaurine, norarmepavine and codeine were tested against the epimastigotes of the Tulahuén and LQ strains and the DM 28c clone of Trypanosoma cruzi. The micromolar concentration to inhibit 50% of the culture growth (Tulahuén strain) for apomorphine, glaucine, predicentrine, boldine, norarmepavine, coclaurine and codeine were 29, 90, 85, 110, 310, 580 and > 1000 respectively. Similar values were obtained with the LQ strain and the DM 28c clone. The most active compounds in inhibiting culture growth also inhibited cell respiration, suggesting that these drugs may act by blocking mitochondrial electron transport. The trypanocidal effects of these alkaloids appear to be correlated with their antioxidative activities.

Effects of 2(3)-tert-butyl-4-hydroxyanisole (BHA) on in situ mitochondria of Trypanosoma cruzi

Results obtained with in situ mitochondria or Trypanosoma cruzi showed that this protozoon had only two energy coupling sites, sites II and III that correspond to higher eukaryote mitochondria. Rotenone did not inhibit the oxygen uptake or the parasite. These results suggest that the NADH‐ubiquinone segment of the respiratory chain has no activity. Studies with in situ mitochondria confirmed that BHA, an antioxidant food additive, blocks the mitochondrial electron transport chain at the succinate‐cytochrome b segment being the molecular basis of this trypanocidal action.

Trypanosoma cruzi: carboxylesterase activity in intact epimastigotes.

Carboxylesterase activity corresponding to types A and B has been demonstrated in intact T. cruzi epimastigotes as shown by the hydrolysis of several esters of p-nitrophenol and the effect of suitable inhibitors. The in situ carboxylesterase activity was described by the Michaelis Menten kinetic approach. The apparent Vmax for the acetate and butyrate esters were 66.5 and 165.3 nmol hydrolysed per min and mg of protein respectively. An Arrhenius plot of the temperature dependent activity showed two sharp linear regions with a transition temperature of 31.6 degrees C. and energies of activation of 6.2 and 14.1 kcal/mol. The in situ carboxylesterase activity was inhibited 26% by paraoxon and 56% by N-ethylmaleimide, but not by p-chloromercuribenzoate.

Nitro radical anion formation from nifurtimox II: electrochemical evidence

Abstract The cyclic voltammetric (CV) behaviour of nifurtimox was studied. The addition of aprotic solvent (DMF) to nifurtimox in an aqueous citrate buffer system was examined. Qualitatively this results in separation of the initial irreversible four-electron reduction into two stages, the RNO 2 /RNO 2 .− and RNO 2 .− ,4H + /RNHOH,H 2 O couples respectively. Particul attention was directed to the one-electron RNO 2 /RNO 2 .− couple as measured by the CV mode in mixed media. Analysis of the CV response as a function scan rate and non-aqueous solvent content yields information on the stability of the radical anion. The chemical forward reaction of the radical anion follows a second-order kinetic with a stability constant of 954 L mol −1 s −1 and a half-life of 1.05 s for 1 mM nifurtimox in aqueous citrate buffer, pH 10.5, + DMF, 40:60.

Effects of hydroquinones on intact Trypanosoma cruzi epimastigotes

1. Hydroquinones inhibited the culture growth of Trypanosoma cruzi epimastigotes at concentrations lower than 1 mM. 2. Hydroquinones inhibited the oxygen consumption on the intact Trypanosoma cruzi cells. I50 values for hydroquinone, terbutylhydroquinone and 2,5-di-t-butylhydroquinone were 24.87 mM, 0.88 mM and 0.26 mM, respectively. t-Butylhydroquinone and 2,5-di-t-butylhydroquinone had a Michaelian type kinetic inhibition; hydroquinone also showed a Michaelian type kinetic inhibition at low concentrations but at higher concentrations it showed a positive cooperativity. 3. These hydroquinones changed the NAD(P) redox-state to a more reduced state and that of cytochrome b to a more oxidized state. The magnitude of the redox-state change was dependent of the hydrophobicity of the derivates. 4. These results suggest that the growth and oxygen uptake inhibition by the hydroquinones is due to a blockage of the mitochondrial electron transport chain before cytochrome b.

The γ-glutamyltranspeptidase of Trypanosoma cruzi

1. 1. Trypanosoma cruzi epimastigotes show γ-glutamyltranspeptidase activity which has characteristics significantly different than the mammalian enzyme. 2. 2. The protozoan enzyme is localized in the cytosolic fraction, it has a Km of 1.6 mM and a Vmax of 17.4 nmol/min/mg protein with l-γ-glutamyl-p-nitroanilide as γ-glutamyl donor, and an optimun pH range from 7.5 to 8.0. 3. 3. The best amino acid acceptors were l-histidine, l-asparagine, l-aspartate, l-glutamate and l-proline, but l-glutamine was a very poor acceptor. 4. 4. The enzyme was very sensitive to inhibition by 6-diazo-5-oxo-l-norleucine (k2 = 4.0 × 105M per min) and O-diazo-acetyl-l-serine (k2 = 1.1 × 104M per min). Phenobarbital (k2 = 8.38M per min) and l-serine borate (Ki = 34 mM) were poor inhibitors. 5. 5. The activity of the enzyme was not correlated with the logarithmic phase of growth of the parasites and steadily decreases with the age of the cultures.

Nitro Aryl 1,4-Dihydropyridine Derivatives: Effects on Trypanosoma cruzi

A series of nitro aryl 1,4-dihydropyridine derivatives produced inhibition of both cell growth and oxygen consumption on Tulahuen and LQ strains, and clone Dm 28c of epimastigotes of Trypanosoma cruzi. Nicardipine was found to be the most potent derivative in both growth cell (I50 = 70 microM) and oxygen uptake (I50 = 26 microM in intact parasites, I50 = 325 microM in situ mitochondria). A correlation between the inhibitory effects on the growth cell and the apparent first order kinetic for the uptake of the 1,4-dihypyridine derivatives by T. cruzi epimastigotes was found. Thus, nicardipine, the most potent derivative, exhibited the highest apparent rate constant, ku, (0.043 min-1). On the other hand, no susceptibility differences by strains and clone Dm 28c to the action of these drugs were found.

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds☆

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.