Potential association of Trypanosoma cruzi DTUs TcV and TcVI with the digestive form of Chagas disease.

The relationship among genetic diversity of Trypanosoma cruzi and clinical forms of Chagas disease remain elusive. In order to assess the possible association between different T. cruzi Discrete Typing Units (DTUs) and the clinical pictures of the disease, 205 chronic patients from Salta province, Argentina, were analysed. One hundred and twenty-two of these patients were clinically categorized as: cardiac 38.5% (47/122), digestive 15% (18/122), cardio-digestive 16% (20/122) and asymptomatic 30% (37/122). From each patient, blood samples were taken for both, Polymerase Chain Reaction (PCR) targeting kDNA and blood culture analyses. The presence of T. cruzi kDNA was detected in 43% (88/205) of the patients. T. cruzi DTUs were identified in 74% (65/88) of the kDNA positive patients by PCR-hybridization using specific probes. We detected the presence of DTUs TcI, TcII, TcV and TcVI. Single infections (i.e. presence of only one DTU in the sample) were detected in 38.64% of the samples (34/88), while mixed infections were 35.23% (31/88). TcV was the most prevalent DTU (60.3%- 53/88). The association analyses showed, for the first time to the best of our knowledge, that TcV and TcVI were associated with the digestive form of Chagas Disease (Fisher p = .0001).

Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence.

Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites' antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson's coefficient: 0.617, 0.771, 0.499; respectively, P<0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease.

The antioxidant properties of zinc: interactions with iron and antioxidants.

Potential mechanisms underlying zinc's capacity to protect membranes from lipid oxidation were examined in liposomes. Using lipid oxidation initiators with different chemical and physical properties (transition metals, lipid- or water-soluble azo compounds, ultraviolet radiation c (UVc), superoxide radical anion (O2*-), and peroxynitrite (ONOO-) we observed that zinc only prevented copper (Cu2+)- and iron (Fe2+)-initiated lipid oxidation. In the presence of Fe2+, the antioxidant action of zinc depended directly on the negative charge density of the membrane bilayer. An inverse correlation (r2: 0.96) was observed between the capacity of zinc to prevent iron binding to the membrane and the inhibitory effect of zinc on Fe2+-initiated lipid oxidation. The interaction of zinc with the bilayer did not affect physical properties of the membrane, including rigidification and lateral phase separation known to increase lipid oxidation rates. The interactions between zinc and the lipid- (alpha-tocopherol) and water- (epicatechin) soluble antioxidants were studied. The inhibition of Fe2+-induced lipid oxidation by either alpha-tocopherol or epicatechin was increased by the simultaneous addition of zinc. The combined actions of alpha-tocopherol (0.01 mol%), epicatechin (0.5 microM) and zinc (5-50 microM) almost completely prevented Fe2+ (25 microM)-initiated lipid oxidation. These results show that zinc can protect membranes from iron-initiated lipid oxidation by occupying negatively charged sites with potential iron binding capacity. In addition, the synergistic actions of zinc with lipid and water-soluble antioxidants to prevent lipid oxidation, suggests that zinc is a pivotal component of the antioxidant defense network that protects membranes from oxidation.

Zinc deficiency induces oxidative stress and AP-1 activation in 3T3 cells.

It has been postulated that one mechanism underlying zinc deficiency-induced tissue alterations is excessive cellular oxidative damage. In the present study we investigated if zinc deficiency can induce oxidative stress in 3T3 cells and trigger select intracellular responses that have been associated to oxidative stress. Cells were exposed to control media or to chelated media containing 0.5, 5, or 50 microM zinc for 24 or 48 h. The oxidative status of the cells was evaluated as an increase in the fluorescence of the probe 5(or 6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate (DCDCDHF). After 24 and 48 h of exposure, the fluorescence intensity was significantly higher (4- to 15-fold) in the 0.5 and 5 microM Zn groups compared to the 50 microM Zn and control groups. The activity of the antioxidant enzymes CuZn (CuZnSOD) and Mn (MnSOD) superoxide dismutases was significantly higher in the 0.5 and 5 microM Zn cells compared to the 50 microM Zn and control groups at both the 24 and 48 h time points. These higher activities were associated with higher levels of MnSOD mRNA. After 24 h in culture, the level of activated AP-1 was markedly higher in the 0.5 and 5 microM Zn cells than in the control (72 and 58%, respectively) and 50 microM Zn cells (73 and 60%, respectively). NF-kappaB binding activity was lower in the 0.5 and 5 microM Zn cells than in controls. Thus, oxidative stress is induced by zinc deficiency in 3T3 cells. This oxidative stress results in an upregulation of oxidant defense mechanisms.

Zinc in the prevention of Fe2+-initiated lipid and protein oxidation.

In the present study we characterized the capacity of zinc to protect lipids and proteins from Fe2+-initiated oxidative damage. The effects of zinc on lipid oxidation were investigated in liposomes composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS) at a molar relationship of 60:40 (PC:PS, 60:40). Lipid oxidation was evaluated as the oxidation of cis-parinaric acid or as the formation of 2-thiobarbituric acid-reactive substances (TBARS). Zinc protected liposomes from Fe2+ (2.5-50 microM)-supported lipid oxidation. However, zinc (50 microM) did not prevent the oxidative inactivation of glutamine synthetase and glucose 6-phosphate dehydrogenase when rat brain supernatants were oxidized in the presence of 5 microM Fe2+ and 0.5 mM H2O2. We also studied the interactions of zinc with epicatechin in the prevention of lipid oxidation in liposomes. The simultaneous addition of 0.5 microM epicatechin (EC) and 50 microM zinc increased the protection of liposomes from oxidation compared to that observed in the presence of zinc or EC separately. Zinc (50 microM) also protected liposomes from the stimulatory effect of aluminum on Fe2+-initiated lipid oxidation. Zinc could play an important role as an antioxidant in biological systems, replacing iron and other metals with pro-oxidant activity from binding sites and interacting with other components of the oxidant defense system.

Zinc in the prevention of Fe2+-initiated lipid and protein oxidation

In the present study we characterized the capacity of zinc to protect lipids and proteins from Fe2+-initiated oxidative damage. The effects of zinc on lipid oxidation were investigated in liposomes composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS) at a molar relationship of 60:40 (PC:PS, 60:40). Lipid oxidation was evaluated as the oxidation of cis-parinaric acid or as the formation of 2-thiobarbituric acid-reactive substances (TBARS). Zinc protected liposomes from Fe2+ (2.5-50 microM)-supported lipid oxidation. However, zinc (50 microM) did not prevent the oxidative inactivation of glutamine synthetase and glucose 6-phosphate dehydrogenase when rat brain supernatants were oxidized in the presence of 5 microM Fe2+ and 0.5 mM H2O2. We also studied the interactions of zinc with epicatechin in the prevention of lipid oxidation in liposomes. The simultaneous addition of 0.5 microM epicatechin (EC) and 50 microM zinc increased the protection of liposomes from oxidation compared to that observed in the presence of zinc or EC separately. Zinc (50 microM) also protected liposomes from the stimulatory effect of aluminum on Fe2+-initiated lipid oxidation. Zinc could play an important role as an antioxidant in biological systems, replacing iron and other metals with pro-oxidant activity from binding sites and interacting with other components of the oxidant defense system.