Evaluating the effect of curcumin on the metacestode of Taenia crassiceps.

Curcumin, a curcuminoid present in the rhizome of the plant Curcuma longa has multiple pharmacological effects including anticarcinogenic and anti-inflammatory properties. This work evaluates the anthelmintic effect of the curcumin molecule (98% pure) on Taenia crassiceps cysticerci viability in vitro. Cysticerci incubated in the presence of increasing concentrations of curcumin showed a dose-dependent mortality correlated with a significant increase in the production of reactive oxygen species and a partial inhibition of thioredoxin-glutathione reductase, the only disulfide reductase present in these parasites. At 500 µM curcumin, a 100% of cysticerci lethality was obtained after 2 h of treatment. These results suggest the curcumin-induced oxidative stress could be in the origin of the anthelminthic effect of curcumin. Mice with cysticerci were injected intraperitoneally with 20, 40, or 60 mM curcumin daily for 30 days. A decrease in the burden of cysticerci (46%) was observed with a 60 mM dose of curcumin, supporting this compound as a potential anthelmintic drug.

Evolutionary Adaptations of Parasitic Flatworms to Different Oxygen Tensions.

During the evolution of the Earth, the increase in the atmospheric concentration of oxygen gave rise to the development of organisms with aerobic metabolism, which utilized this molecule as the ultimate electron acceptor, whereas other organisms maintained an anaerobic metabolism. Platyhelminthes exhibit both aerobic and anaerobic metabolism depending on the availability of oxygen in their environment and/or due to differential oxygen tensions during certain stages of their life cycle. As these organisms do not have a circulatory system, gas exchange occurs by the passive diffusion through their body wall. Consequently, the flatworms developed several adaptations related to the oxygen gradient that is established between the aerobic tegument and the cellular parenchyma that is mostly anaerobic. Because of the aerobic metabolism, hydrogen peroxide (H2O2) is produced in abundance. Catalase usually scavenges H2O2 in mammals; however, this enzyme is absent in parasitic platyhelminths. Thus, the architecture of the antioxidant systems is different, depending primarily on the superoxide dismutase, glutathione peroxidase, and peroxiredoxin enzymes represented mainly in the tegument. Here, we discuss the adaptations that parasitic flatworms have developed to be able to transit from the different metabolic conditions to those they are exposed to during their life cycle.

Correction: Effect of curcuminoids and curcumin derivate products on thioredoxin-glutathione reductase from Taenia crassiceps cysticerci. Evidence suggesting a curcumin oxidation product as a suitable inhibitor.

[This corrects the article DOI: 10.1371/journal.pone.0220098.].

Effect of curcuminoids and curcumin derivate products on thioredoxin-glutathione reductase from Taenia crassiceps cysticerci. Evidence suggesting a curcumin oxidation product as a suitable inhibitor.

Curcuma is a traditional ingredient of some Eastern cuisines, and the spice is heralded for its antitumoral and antiparasitic properties. In this report, we examine the effect of the curcuminoides which include curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), as well as curcumin degradation products on thioredoxin glutathione reductase from Taenia crassiceps cysticerci Results revealed that both DMC and BDMC were inhibitors of TGR activity in the micromolar concentration range. By contrast, the inhibitory ability of curcumin was a time-dependent process. Kinetic and spectroscopical evidence suggests that an intermediary compound of curcumin oxidation, probably spiroepoxide, is responsible. Preincubation of curcumin in the presence of NADPH, but not glutathione disulfide (GSSG), resulted in the loss of its inhibitory ability, suggesting a reductive stabilizing effect. Similarly, preincubation of curcumin with sulfhydryl compounds fully protected the enzyme from inhibition. Degradation products were tested for their inhibitory potential, and 4-vinylguaiacol was the best inhibitor (IC50 = 12.9 µM), followed by feruloylmethane (IC50 = 122 µM), vanillin (IC50 = 127 µM), and ferulic aldehyde (IC50 = 180 µM). The acid derivatives ferulic acid (IC50 = 465 µM) and vanillic acid (IC50 = 657 µM) were poor inhibitors. On the other hand, results from docking analysis revealed a common binding site on the enzyme for all the compounds, albeit interacting with different amino acid residues. Dissociation constants obtained from the docking were in accord with the inhibitory efficiency of the curcumin degradation products.

Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR).

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala).

A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

The Architecture of Thiol Antioxidant Systems among Invertebrate Parasites.

The use of oxygen as the final electron acceptor in aerobic organisms results in an improvement in the energy metabolism. However, as a byproduct of the aerobic metabolism, reactive oxygen species are produced, leaving to the potential risk of an oxidative stress. To contend with such harmful compounds, living organisms have evolved antioxidant strategies. In this sense, the thiol-dependent antioxidant defense systems play a central role. In all cases, cysteine constitutes the major building block on which such systems are constructed, being present in redox substrates such as glutathione, thioredoxin, and trypanothione, as well as at the catalytic site of a variety of reductases and peroxidases. In some cases, the related selenocysteine was incorporated at selected proteins. In invertebrate parasites, antioxidant systems have evolved in a diversity of both substrates and enzymes, representing a potential area in the design of anti-parasite strategies. The present review focus on the organization of the thiol-based antioxidant systems in invertebrate parasites. Differences between these taxa and its final mammal host is stressed. An understanding of the antioxidant defense mechanisms in this kind of parasites, as well as their interactions with the specific host is crucial in the design of drugs targeting these organisms.