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Comet assay method for DNA damage "measurement" caused by photoinduced oxidative stress in cells

Grant number: 18/06301-6
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): April 01, 2018
Effective date (End): March 31, 2019
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Paolo Di Mascio
Grantee:Bruna Dias Carvalho da Costa
Home 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 by 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. 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 pirimidines respectively, generating strand breaks that are detected by electrophoresis. (AU)