Biochemical and microscopy evidence on adverse effects of nitroxidized human serum albumin

Abstract

Earlier researches have pointed about the accumulation of peroxynitrite modified proteins and their aggregates in the etiopathogenesis of many age-related neurodegenerative and several autoimmune diseases. Human serum albumin (HSA) is present in abundance in plasma and is susceptible to modification by peroxynitrite. In this study, HSA modified with peroxynitrite (nitroxidized-HSA) formed aggregate besides other gross structural changes. Aggregation or assembly of aberrant proteins is responsible for increase in production of reactive species and is often correlated with toxicity in neurodegenerative diseases. However, lack of literature on the cytotoxicity of aggregated nitroxidized-HSA led us to explore its toxicity using human peripheral blood lymphocytes. Elevated protein carbonyl coupled with decreased protein thiol, and release of antioxidant enzymes and lactate dehydrogenase (LDH) were observed upon incubation of lymphocytes with nitroxidized-HSA. Trypan blue exclusion and MTT assays indicated nitroxidized-HSA induced injury/death of lymphocytes. This may be attributed to the observed reactive oxygen species generation during the interaction of nitroxidized-HSA with lymphocytes. Moreover, the analysis of the cellular morphology by dual staining, fluorescence and confocal microscopy further confirms the cytotoxicity of nitroxidized-HSA. Since various age-related degenerative diseases are characterized by deposition of protein aggregates, the demonstrated toxicity of nitroxidized-HSA may be an important driver in the pathophysiology of neurodegenerative diseases.