Role of nitric oxide and superoxide in Giardia lamblia killing

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (~60% dead trophozoites). This effect was inhibited (>90%) by an NO synthase inhibitor (200 µM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 µM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (~35% dead trophozoites at 1 mM). The mixture of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoite killing and this effect is not mediated by peroxynitrite

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (~40%) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.