Singlet molecular oxygen produced by UVA irradiation after incorporation of 6-Thioguanine into DNA

• Grant number: 22/03833-2
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: April 01, 2023
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Biophysics - Radiology and Photobiology
• Principal Investigator: Paolo Di Mascio
• Grantee: André Luiz Lopes
• 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

Singlet molecular oxygen (1O2) has been shown to be generated in biological systems. Biological targets for 1O2 include unsaturated fatty acids, proteins, and DNA. Preliminary results obtained in an undergraduate research project showed that 0.2 to 4 % of 6-thioguanine (6-TG) is incorporated in the DNA of HaCat epithelial cells. In this view, this research project aims to use 6-thioguanine (6-TG) as a photosensitizer molecule to epithelial cells. This project will expand the study using different epithelial cells, such as NHEK keratinocytes and CCD-1059Sk and HDFa fibroblasts. Once 6-TG is incorporated in the DNA, the cells will be irradiated with UVA, and we will test whether or not 6-TG will give rise to the production of 1O2 in situ in the cells' DNA. The purpose of using 6-TG as a 1O2 generator in cells is to understand the oxidative damage mechanisms caused by singlet oxygen in a complex cellular system. With that in mind, this work will seek for 1O2-oxidative signatures in DNA and nuclear membrane of cells. Oxidation products, such as 8-oxoguanine, will be detected and quantified by comet assay and mass spectrometry. Another goal is to verify the extension of 1O2-induced damage by quantification of the 5±-OOH cholesterol hydroperoxide (a specific marker of 1O2 oxidation) in nuclear membrane, also using mass spectrometry. Through this work, we aim to unravel the mechanisms of damage caused by 1O2 in cells, especially those submitted to UVA radiation. Our work may help in the understanding of pathologies triggered by exposition to this oxidant. (AU)