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Comet Assay: Detection of Damage Induced by UVA Light After Incorporation of 6-Thioguanine into the DNA of Human Keratinocytes

Grant number: 23/12607-9
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): February 01, 2024
Effective date (End): January 31, 2025
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Paolo Di Mascio
Grantee:Thabata Victória Ferreira
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

Even though the solar light is crucial to life, playing major roles in human physiology, it is also one of the most important and ubiquitous carcinogens to which humans are exposed. The ultraviolet (UV) component emitted by the sun is composed of UVC, UVB and UVA. However, UVC is completely absorbed by the atmosphere, making UVA and UVB wavelengths of major relevance for human health. UV light affects mainly tissues exposed to the environment, as the skin. For a long time, the deleterious effects of UVA light exposure were neglected, however, UVA radiation can be absorbed by cellular chromophores, leading to the formation of photoexcited species, such as singlet oxygen, that can act as cellular stress mediators or signaling molecules.This research project aims to use 6-thioguanine (6-TG) as a photosensitizer molecule to epithelial cells. Once 6-TG is incorporated in the DNA, HaCat 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.Comet assay or Single Cell Gel Electrophoresis is a versatile and sensitive method for measuring single (SSB) and double-strand breaks (DSB) in DNA. Comets are formed by the relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break release, allowing this loop to migrate to the anode under electrophoretic condition. Besides its ability to detect SSB and DSB, the comet assay can also be used to measure oxidized bases. This is achieved with an additional step that relies on the addition of bacterial endonucleases after cell lysis; these enzymes are able to generate a strand break where an oxidative modification took place. These enzymes, oxoguanine glycosylase (OGG1) and endonuclease V (T4endoV), recognizes oxidized purines and pyrimidines respectively, generating strand breaks that are detected by electrophoresis.

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