Formation of nitrosyl hemoglobin and nitrotyrosine during murine leishmaniasis

Peroxynitrite, the potent oxidant formed by the fast reaction between nitric oxide and superoxide anion, has been suggested to be the reactive intermediate responsible for some of the pathologies associated with an over-production of nitric oxide. In this report, we demonstrate that both nitric oxide and peroxynitrite are formed during infection of the susceptible mouse strain, BALB/c, with Leishmania amazonensis. Nitric oxide was detected as the nitrosyl hemoglobin complex by EPR analysis of blood drawn from mice at 35, 64 and 148 days of infection. The levels of nitrosyl hemoglobin complex increased with disease evolution, which in the murine model used is characterized by skin lesions, ulceration and visceralization of the parasites. Peroxynitrite formation was inferred from immunoreaction of homogenates obtained from footpad lesions in the late stages of the infection with anti-nitrotyrosine antibody; homogenates from parasites drawn from the lesions were also immunoreactive, although to a lesser extent. Analysis of protein homogenates by gel electrophoresis and western blots suggests that peroxynitrite may degrade proteins in vivo, in addition to nitrating them. The results demonstrate that peroxynitrite is formed during murine leishmaniasis and may play a role in the aggravation of the disease.