Detection and analysis of singlet oxygen mechanisms in leukocytes

• Grant number: 21/08842-7
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: September 01, 2022
• End date: July 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Graziella Eliza Ronsein
• Grantee: Rafaela Oliveira Nascimento
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

Neutrophils and eosinophils are key leukocytes for the immune system. Neutrophils are the first cells that are recruited to inflammatory sites and are capable of orchestrate the immune response. Eosinophils also kill pathogens, mainly fungi and parasites. Moreover, they are associated with diseases such as Asthma, Rhinitis, and gastrointestinal infections. Both eosinophils and neutrophils show many defense mechanisms that can involve reactive oxygen species generation. One species possibly involved in this mechanism is singlet oxygen (1O2), a potent oxidant in the organism. However, its presence and formation mechanisms in leukocytes are contradictory. Thus, this work aims to 1) analyze the 1O2 formation in activated leukocytes. The experiments will be performed using neutrophil (HL-60) and eosinophil (EoL-1) cell lines. The results will be confirmed using neutrophil and eosinophil isolated from peripheric blood; 2) investigate the role of different reactive species present in leukocytes in 1O2 generation, for example, reactions involving haloamines. To detect 1O2, we will use highly specific methods by monitoring the generation of light in the near-infrared region and using the singlet oxygen chemical trap anthracene-9,10-divinylsulfonate (AVS) analyzed by HPLC/MS/MS analysis; 3) previous results obtained in our work indicated by HPLC/MS/MS analysis that methionine reaction with oxidants present in leukocytes (HOCl, HOBr and 1O2) can generate specific products as markers of these oxidants in the cells. Now we aim to analyze the reactions mechanisms of peptides and proteins containing methionine in the N-terminal, with the purpose to identify products that can act as specific signatures of oxidants present in leukocytes. (AU)