M. Paola Zago

The antioxidant properties of zinc: Interactions with iron and antioxidants

Potential mechanisms underlying zincs 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 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.

α-Lipoic acid and N-acetyl cysteine prevent zinc deficiency-induced activation of NF-κB and AP-1 transcription factors in human neuroblastoma IMR-32 cells

This work investigated the capacity of α-lipoic acid (LA) and N-acetyl-l-cysteine (NAC) to reduce zinc deficiency-induced oxidative stress, and prevent the activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and the cross-talk between both activated cascades through β-Transducin Repeat-containing Protein (β-TrCP). IMR-32 cells were incubated in control media or media containing variable concentrations of zinc, without or with 0.5 mM LA or 1 mM NAC. Relative to control and zinc supplemented (15 μM Zn) groups, Hydrogen peroxide (H2O2) and total oxidant cell concentrations were higher, and total glutathione concentrations were lower in the zinc deficient groups (1.5 and 5 μM Zn). Both, LA and NAC, markedly reduced the increase in cell oxidants and the reduction in glutathione concentrations in the zinc deficient cells. Consistent with this, LA and NAC prevented zinc deficiency-induced activation of the early steps of NF- κB (IκBα phosphorylation) and AP-1 [c-Jun-N-terminal kinase (JNK) and p38 phophorylation] cascades, and the high NF-κB- and AP-1-DNA binding activities in total cell extracts. Thus, LA and NAC can reduce the oxidative stress associated with zinc deficiency and the subsequent triggering of NF-κB- and AP-1-activation in neuronal cells.

Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution.

Zinc Deficiency in Neuronal Biology

Adverse nutritional and environmental conditions during early development can irreversibly affect the nervous system. Zinc (Zn) deficiency associated with inadequate Zn intake and undernutrition is frequent throughout the world. Increasing evidence indicates that developmental Zn deficiency can lead to alterations in neonate and infant behavior, cognitive and motor performance that persist into adulthood. This review will address current knowledge on the events that are triggered in neuronal cells when Zn availability decreases and discuss their consequences on neuronal function and development. In neuronal cells, Zn deficiency induces oxidative stress, alters the normal structure and dynamics of the cytoskeleton, affects the modulation of transcription factors AP‐1, NF‐βB and NFAT and induces a decreased cell proliferation and increased apoptotic death. Thus, these closely associated events can affect neuronal function and critical developmental events (neuronal proliferation, differentiation, plasticity and survival) when Zn availability decreases. IUBMB Life, 59: 299‐307, 2007

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi

Background: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione‐spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol‐redox metabolite in trypanosomatids. Methods and results: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti‐protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36–71% higher resistance to H2O2 (50–1000 &mgr;M) and heavy metal (1–500 &mgr;M) toxicity. Treatment with TryS inhibitors (5–30 &mgr;M) abolished the proliferation and survival advantages against H2O2 pressure in a dose‐dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. Conclusions: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti‐trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease. Graphical abstract Figure. No Caption available. HighlightsTrypanothione synthetase (TryS) is an essential enzyme for T. cruzi.TryS‐overexpression confers higher replication rate to T. cruzi epimastigotes.TryS provides protection against oxidative stress to different parasite stages.TryS confers resistance to current treatment drugs, benznidazole and nifurtimox.