Phagocytic uptake of oxidized heme polymer is highly cytotoxic to macrophages.

Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or ß-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. ß-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of ß-hematin is observed in macrophages treated with ß-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of ß-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes ß-hematin to form oxidized ß-hematin (ßH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the ß-hematin toxicity, indicates crucial role of ßH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

Methemoglobin exposure produces toxicological effects in macrophages due to multiple ROS spike induced apoptosis.

Macrophages are an integral part of the immune system, required to produce a robust immune response against an infectious organism. Presence of methemoglobin in body fluids such as blood, cerebrospinal fluid and urine is associated with tissue damage. We tested cytotoxic effects of MetHb and underlying molecular events in mouse macrophage cell line J774A.1. MetHb exposure dose dependently reduced macrophage viability in an MTT assay. Light microscopy and scanning electron microscopic (SEM) observation of MetHb treated macrophage indicated death (less number of cells per field), severe damage to membrane structure and accumulation of particulate matter in the cytosol. The macrophage death during MetHb exposure was due to induction of apoptosis as indicated by annexin-V/FITC staining and DNA fragmentation analysis. MetHb treatment generated a periodic ROS spikes with time in the macrophage cytosol to develop oxidative stress. Scavenging ROS spikes with NAC, mannitol or PBN dose dependently protected macrophages against MetHb induced toxicity, apoptosis and cellular membrane damage. Our work highlighted the contributions of MetHb mediated toxicity toward macrophage and its potential role in tissue damage and immune depression during malaria and other hemolytic disorders.

Oxido nítrico: fisiología y farmacología / Nitric oxide: physiology and pharmacology

El óxido nítrico pasó de ser una molécula tóxica, producto de la combustión a ser un mediador de múltiples procesos biológicos. Los principales investigadores que trabajaron sobre el mismo recibieron en el año 1998 el premio Nobel. Se lo encuentra presente en las células del endotelio de todos los mamíferos y juega un papel fundamental en el proceso de relajación. Posteriormente se ha demostrado que se sintetiza en las plaquetas, células del sistema inmune, neuronas y otras células del organismo donde ejerce, otras acciones más allá de la vasodilatación. Su exceso o defecto permitió la comprensión de distintos mecanismos fisiopatológicos (shock séptico, hipertensión arterial). Su modulación permite tratar distintas entidades. La administración por vía inhalatoria tiene utilidad en diversas patologías. El tratamiento de la hipertensión pulmonar del recién nacido es el ejemplo más relevante, porque permitió mejorar la sobrevida y disminuyó el número de pacientes sometidos a Membrana de Oxigenación Extracorpórea. En el tratamiento de las cardiopatías congénitas que cursan con hipertensión pulmonar ocupa un lugar destacado en el intra, pre y postoperatorio. El Síndrome de Distrés Respiratorio del Adulto es quizá la entidad más cuestionada; ya que, si bien mejora la hipoxemia, no hay estudios que demuestren que mejora la sobrevida. (AU)

Pathogenetic mechanisms in experimental hemoglobinuric acute renal failure.

To evaluate mechanisms in hemoglobinuric acute renal failure (ARF) rats were infused with hemoglobin under aciduric or alkalinuric conditions. Aciduric rats developed azotemia, distal heme casts, and proximal tubular cell (PTC) necrosis, whereas alkalinuric rats developed no renal damage. Aciduria converted hemoglobin to met-hemoglobin, which precipitated, forming distal casts and inducing ARF. Hematin formation was not observed. The importance of met-hemoglobin production was indicated by its greater toxicity than hemoglobin during aciduria and by its ability to induce ARF even under alkalinuric conditions. A link between obstructing casts and PTC necrosis was identified; tubular obstruction induced by various mechanisms (met-hemoglobin casts; ureteral ligation; ischemic ARF) increased PTC hemoglobin uptake, producing lysosomal overload (giant endolysosomes) and PTC necrosis. This worsened ischemic ARF despite an otherwise subtoxic hemoglobin dose being used that had no discernible acute renal vasoconstrictive effect. Iron chelation (deferoxamine)/hydroxyl radical scavenger (Na benzoate) therapy did not mitigate this exacerbation of ischemic injury, suggesting a nonoxidant mechanism. We conclude that H is nephrotoxic, particularly when intratubular obstruction facilitates PTC heme uptake. Thus aciduria-induced met-hemoglobin cast formation and concomitant ischemic renal injury predispose to its nephrotoxic effect.